Recombinant protective protein from Streptococcus pneumoniae

ABSTRACT

The present invention discloses amino acid sequences and nucleic acid sequences relating to a  Streptococcus Pneumoniae  surface associated Pneumo Protective Protein (PPP) having a molecular weight of about 20 kilo Daltons (kDa). The PPP exhibits the ability to reduce colonization of pneumococcal bacteria. Thus the present invention also pertains to compositions for the treatment and prophylaxis of infection or inflammation associated with bacterial infection.

PRIORITY

This is the U.S. national phase application under 35 U.S.C. §371 ofInternational Patent Application Ser. No. PCT/US01/49650, filed Dec. 28,2001 and published in English on Jul. 11, 2002 under InternationalPublication No. 02/053761, and also claims priority under 35 U.S.C.§119(e) from U.S. Provisional Patent Application Ser. No. 60/258,841,filed Dec. 28, 2000, which are both hereby incorporated by reference intheir entirety.

FIELD OF THE INVENTION

The present invention provides amino acid sequences and nucleic acidsequences relating to a protein of Streptococcus pneumoniae having amolecular weight of 20 kilo Daltons (kDa). The present invention alsopertains to compositions for the treatment and prophylaxis of infectionor inflammation associated with bacterial infection.

BACKGROUND OF THE INVENTION

The middle ear is a sterile, air-filled cavity separated from the outerear by the eardrum. Attached to the eardrum are three ear bones thatvibrate when sound waves strike the eardrum. Vibrations are transmittedto the inner ear, which generates nerve impulses that are sent to thebrain. Air may enter the middle ear through the Eustachian tube, whichopens in the walls of the nasopharynx.

The nasopharynx is located posterior to the nasal cavities. Thenasopharynx is lined by the respiratory epithelium and stratifiedsquamous epithelium. Beneath the respiratory epithelium, the abundantmucosa-associated lymphoid tissue (MALT) forms the nasopharyngeal tonsil(adenoids).

Bacterial infection or inflammation of the middle ear is mainly observedin children. Due to the isolation of the middle ear, it is suggestedthat development of middle ear infections requires the involvement ofthe nasopharynx and Eustachian tube. Infections with Streptococcuspneumoniae (S. pneumoniae) are one of the major causes of middle earinfections, as well as bacteremia, meningitis, and fatal pneumoniaworldwide (Butler, J. C., et al., American Journal of Medicine, 1999,107:69S–76S). The rapid emergence of multi-drug resistant pneumococcalstrains throughout the world has led to increased emphasis on preventionof pneumococcal infections by vaccination (Goldstein and Garau, Lancet,1997, 350:233–4).

Protein antigens of S. pneumoniae have been evaluated for protectiveefficacy in animal models of pneumococcal infection. Some of the mostcommonly studied vaccine candidates include the the PspA proteins, PsaAlipoprotein, and the CbpA protein. Numerous studies have shown that PspAprotein is a virulence factor (Crain, M. J., et al., Infect Immun, 1990,58:3293–9; McDaniel, L. S., et al., J Exp Med,1984, 160:386–97), but isantigenically variable among pneumococcal strains. Additionally, arecent study has indicated that some antigenically conserved regions ofa recombinant PspA variant may elicit cross-reactive antibodies in humanadults (Nabors, G. S., et al., Vaccine, 2000, 18:1743–1754). PsaA, a 37kDa lipoprotein with similarity to other Gram-positive adhesins, isinvolved in manganese transport in pneumococci (Dintilhac, A., et al.,Molecular Microbiology, 1997, 25(4):727–739; Sampson, J. S., et al.,Infect Immun, 1994, 62:319–24.) and has been shown to be protective inmouse models of systemic disease (Talkington, D. F., et al., MicrobPathog, 1996. 21:17–22). The surface exposed choline binding protein,CbpA, is antigenically conserved and also is protective in mouse modelsof pneurnococcal disease (Rosenow, C., et al. Molecular Microbiology,1997, 25:819–29). Since nasopharyngeal colonization is a prerequisitefor otic disease, intranasal immunization of mice with pneumococcalproteins and appropriate mucosal adjuvants has been used to enhance themucosal antibody response and thus, the effectiveness of protein vaccinecandidates (Briles, D. E., et al., Infect Immun, 2000, 68:796–800;Yamamoto, M., et al., A. J Immunol, 1998, 161:4115–21).

The currently available 23-valent pneumococcal capsular polysaccharidevaccine is not effective in children of less than 2 years of age or inimmunocompromised patients, two of the major populations at risk frompneumococcal infection (Douglas, R. M., et al., Journal of InfectiousDiseases, 1983, 148:131–137). A 7-valent pneumococcalpolysaccharide-protein conjugate vaccine, was shown to be highlyeffective in infants and children against systemic pneumococcal diseasecaused by the vaccine serotypes and against cross-reactive capsularserotypes (Shinefield and Black, Pediatr Infect Dis J, 2000, 19:394–7).The seven capsular types cover greater than 80% of the disease isolatesin the United States, but only 57–60% of disease isolates in other areasof the world (Hausdorff, W. P., et al., Clinical Infectious Diseases,2000, 30:100–21). Therefore, there is an immediate need for a vaccine tocover most or all of the disease causing serotypes of pneumococci.

Iron is an essential element for colonization and infection by manypathogenic bacteria. Prevention of the acquisition process should resultin a reduction of colonization and a lower disease potential. Ironacquisition complexes in successful pathogens such as, but not limitedto, N. gonorrheae, N. meningitidis, M. catarrhalis, and H. influenzaehave been evaluated for their vaccine potential by other laboratories(Conte, M. P, et al., Infection and Immunity, 1999, 64:3925; Gray-Owens,S. D., et al. Infection and Immunity, 1995, 64:1201; Luke N. R. et al.,Infection and Immunity, 1999, 67:681; Pettersson, A, et al., Infectionand Immunity, 1993, 61:4724). Thus, isolation of the structuresresponsible for iron acquisition could lead to vaccine candidates.

SUMMARY OF THE INVENTION

The present invention contemplates an isolated S. pneumoniae surfaceassociated Pneumo Protective Protein (PPP) having a molecular weight ofabout 20 kilo Daltons (kDa), where the molecular weight is determinedusing a 10–20% SDS-PAGE gel, or a fragment thereof; the PPP having theability to reduce colonization of pneumococcal bacteria.

The present invention contemplates a recombinant S. pneumoniae surfaceassociated PPP having a molecular weight of about 20 kDa, where themolecular weight is determined using a 10–20% SDS-PAGE gel, or afragment thereof; the PPP having the ability to reduce colonization ofpneumococcal bacteria.

The present invention contemplates a recombinant S. pneumoniae surfaceassociated PPP having a molecular weight of about 20 kDa, where themolecular weight is determined using a 10–20% SDS-PAGE gel, or afragment thereof; the PPP having the ability to reduce colonization ofpneumococcal bacteria; where the PPP has an isoelectric point of about4.587.

The present invention contemplates a recombinant S. pneumoniae surfaceassociated PPP having a molecular weight of about 20 kDa, where themolecular weight is determined using a 10–20% SDS-PAGE gel, or afragment thereof; the PPP having the ability to reduce colonization ofpneumococcal bacteria; where the PPP has an isoelectric point of about4.587 and a charge of about −14.214 at pH 7.

The present invention also contemplates an isolated S. pneumoniaesurface associated PPP having a molecular weight of about 20 kDa, wherethe molecular weight is determined using a 10–20% SDS-PAGE gel, or afragment thereof; where the PPP has an amino acid sequence as depictedin SEQ ID NO: 5, or a fragment thereof; the PPP having the ability toreduce colonization of pneumococcal bacteria.

The present invention also contemplates a nucleic acid sequence encodingan isolated S. pneumoniae surface associated PPP having a molecularweight of about 20 kDa, where the molecular weight is determined using a10–20% SDS-PAGE gel, or a fragment thereof; where the nucleic acidsequence has a sequence as depicted in SEQ ID NO: 4, or a fragmentthereof; the PPP having the ability to reduce colonization ofpneumococcal bacteria.

The present invention also contemplates a cDNA encoding an isolated S.pneumoniae surface associated PPP having a molecular weight of about 20kDa, where the molecular weight is determined using a 10–20% SDS-PAGEgel, or a fragment thereof; where the nucleic acid sequence has asequence as depicted in SEQ ID NO: 4, or a fragment thereof; the PPPhaving the ability to reduce colonization of pneumococcal bacteria.

The present invention contemplates an expression vector comprising anucleic acid sequence encoding an isolated S. pneumoniae surfaceassociated PPP having a molecular weight of about 20 kDa, where themolecular weight is determined using a 10–20% SDS-PAGE gel, or afragment thereof; the PPP having the ability to reduce colonization ofpneumococcal bacteria, where the sequence is operatively associated withan expression control sequence.

The present invention also contemplates a vector comprising a nucleicacid sequence encoding an isolated S. pneumoniae surface associated PPPhaving a molecular weight of about 20 kDa, where the molecular weight isdetermined using a 10–20% SDS-PAGE gel, or a fragment thereof; the PPPhaving the ability to reduce colonization of pneumococcal bacteria,where the sequence is operatively associated with an expression controlsequence, and where the PPP has an isoelectric point of about 4.587.

The present invention further contemplates a vector comprising a nucleicacid sequence encoding an isolated S. pneumoniae surface associated PPPhaving a molecular weight of about 20 kDa, where the molecular weight isdetermined using a 10–20% SDS-PAGE gel, or a fragment thereof; the PPPhaving the ability to reduce colonization of pneumococcal bacteria,where the sequence is operatively associated with an expression controlsequence, and where the PPP has an isoelectric point of about 4.587 anda charge of about −14.214 at pH 7.

The present invention also contemplates an expression vector comprisinga nucleic acid sequence encoding a an isolated S. pneumoniae surfaceassociated PPP having a molecular weight of about 20 kDa, where themolecular weight is determined using a 10–20% SDS-PAGE gel, or afragment thereof; where the PPP has an amino acid sequence as depictedin SEQ ID NO: 5, or a fragment thereof; and where the nucleic acidsequence is operatively associated with an expression control sequence.

The present invention also contemplates an expression vector comprisinga nucleic acid sequence encoding a an isolated S. pneumoniae surfaceassociated PPP having a molecular weight of about 20 kDa, where themolecular weight is determined using a 10–20% SDS-PAGE gel, or afragment thereof; where the PPP has an amino acid sequence as depictedin SEQ ID NO: 5, or a fragment thereof; where the amino acid sequence isencoded by the nucleic acid sequence as depicted in SEQ ID NO: 4, or afragment thereof; and where the nucleic acid sequence is operativelyassociated with an expression control sequence.

The present invention contemplates a host cell transfected with anexpression vector comprising a nucleic acid sequence encoding anisolated S. pneumoniae surface associated PPP having a molecular weightof about 20 kDa, where the molecular weight is determined using a 10–20%SDS-PAGE gel, or a fragment thereof; the PPP having the ability toreduce colonization of pneumococcal bacteria; where the sequence isoperatively associated with an expression control sequence.

The present invention further contemplates a host cell transfected witha vector comprising a nucleic acid sequence encoding an isolated S.pneumoniae surface associated PPP having a molecular weight of about 20kDa, where the molecular weight is determined using a 10–20% SDS-PAGEgel, or a fragment thereof; where the PPP has an amino acid sequence asdepicted in SEQ ID NO: 5, or a fragment thereof; the PPP having theability to reduce colonization of pneumococcal bacteria; where thesequence is operatively associated with an expression control sequence.

The present invention also contemplates a method for producingrecombinant PPP, which method comprises isolating the PPP produced by ahost cell transfected with an expression vector and cultured underconditions that provide for expression of the PPP by the vector, wherethe vector comprises a nucleic acid sequence encoding an isolated S.pneumoniae surface associated PPP having a molecular weight of about 20kDa, where the molecular weight is determined using a 10–20% SDS-PAGEgel, or a fragment thereof; the PPP having the ability to reducecolonization of pneumococcal bacteria; where the sequence is operativelyassociated with an expression control sequence.

The present invention also contemplates a method for producingrecombinant PPP, which method comprises isolating the PPP produced byhost cell transfected with a vector and cultured under conditions thatprovide for expression of the PPP by the vector, where the vectorcomprises a nucleic acid sequence encoding an isolated S. pneumoniaesurface associated PPP having a molecular weight of about 20 kDa, wherethe molecular weight is determined using a 10–20% SDS-PAGE gel, or afragment thereof where the PPP has an amino acid sequence as depicted inSEQ ID NO: 5, or a fragment thereof; the PPP having the ability toreduce colonization of pneumococcal bacteria, where the sequence isoperatively associated with an expression control sequence.

The present invention also contemplates a composition comprising (1) anisolated S. pneumoniae surface associated PPP having a molecular weightof about 20 kDa, where the molecular weight is determined using a 10–20%SDS-PAGE gel, or a fragment thereof; the PPP having the ability toreduce colonization of pneumococcal bacteria; and (2) a pharmaceuticallyacceptable carrier.

The present invention also contemplates a composition comprising (1) anisolated S. pneumoniae surface associated PPP having a molecular weightof about 20 kDa, where the molecular weight is determined using a 10–20%SDS-PAGE gel, or a fragment thereof; the PPP having the ability toreduce colonization of pneumococcal bacteria, and which PPP has an aminoacid sequence as depicted in SEQ ID NO: 5, or a fragment thereof; and(2) a pharmaceutically acceptable carrier.

The present invention contemplates a composition comprising (1) anucleic acid sequence encoding an isolated S. pneumoniae surfaceassociated PPP having a molecular weight of about 20 kDa, where themolecular weight is determined using a 10–20% SDS-PAGE gel, or afragment thereof; the PPP having the ability to reduce colonization ofpneumococcal bacteria, where the nucleic acid sequence has a sequence asdepicted in SEQ ID NO: 4, or a fragment thereof; and (2) apharmaceutically acceptable carrier.

The present invention contemplates a composition comprising (1) anexpression vector comprising a nucleic acid sequence encoding anisolated S. pneumoniae surface associated PPP having a molecular weightof about 20 kDa, where the molecular weight is determined using a 10–20%SDS-PAGE gel, or a fragment thereof; the PPP having the ability toreduce colonization of pneumococcal bacteria, where the sequence isoperatively associated with an expression control sequence; and (2) apharmaceutically acceptable carrier.

The present invention also contemplates a composition comprising (1) anexpression vector comprising a nucleic acid sequence encoding a anisolated S. pneumoniae surface associated PPP having a molecular weightof about 20 kDa, where the molecular weight is determined using a 10–20%SDS-PAGE gel, or a fragment thereof; where the PPP has an amino acidsequence as depicted in SEQ ID NO: 5, or a fragment thereof, and wherethe nucleic acid sequence is operatively associated with an expressioncontrol sequence; and (2) a pharmaceutically acceptable carrier.

The present invention also contemplates a composition comprising (1) ahost cell transfected with an expression vector comprising a nucleicacid sequence encoding an isolated S. pneumoniae surface associated PPPhaving a molecular weight of about 20 kDa, where the molecular weight isdetermined using a 10–20% SDS-PAGE gel, or a fragment thereof; the PPPhaving the ability to reduce colonization of pneumococcal bacteria,where the sequence is operatively associated with an expression controlsequence; and (2) a pharmaceutically acceptable carrier.

The present invention contemplates a composition comprising (1) a hostcell transfected with a vector comprising a nucleic acid sequenceencoding an isolated S. pneumoniae surface associated PPP having amolecular weight of about 20 kDa, where the molecular weight isdetermined using a 10–20% SDS-PAGE gel, or a fragment thereof; the PPPhaving the ability to reduce colonization of pneumococcal bacteria,where the sequence is operatively associated with an expression controlsequence; where the PPP has an amino acid sequence as depicted in SEQ IDNO: 5, or a fragment thereof; and a (2) pharmaceutically acceptablecarrier.

The present invention also contemplates an immunogenic compositioncomprising (i) a S. pneumoniae surface associated PPP having a molecularweight of about 20 kDa, where the molecular weight is determined using a10–20% SDS-PAGE gel, or a fragment thereof; (ii) a pharmaceuticallyacceptable carrier; and (iii) optionally at least one adjuvant.

The present invention also contemplates an immunogenic compositioncomprising (i) a S. pneumoniae surface associated PPP having a molecularweight of about 20 kDa, where the molecular weight is determined using a10–20% SDS-PAGE gel, or a fragment thereof, the PPP having anisoelectric point of about 4.587; (ii) a pharmaceutically acceptablecarrier; and (iii) optionally at least one adjuvant.

The present invention also contemplates an immunogenic compositioncomprising (i) a S. pneumoniae surface associated PPP having a molecularweight of about 20 kDa, where the molecular weight is determined using a10–20% SDS-PAGE gel, or a fragment thereof, PPP having an isoelectricpoint of about 4.587 and a charge of about −14.214 at pH 7; (ii) apharmaceutically acceptable carrier; and (iii) optionally at least oneadjuvant.

The present invention also contemplates an immunogenic compositioncomprising (i) a S. pneumoniae surface associated PPP having a molecularweight of about 20 kDa, where the molecular weight is determined using a10–20% SDS-PAGE gel, or a fragment thereof, which PPP has an amino acidsequence as depicted in SEQ ID NO: 5, or an immunogenic fragmentthereof; (ii) a pharmaceutically acceptable carrier; and (iii)optionally at least one adjuvant.

The present invention also contemplates an immunogenic compositioncomprising (i) a S. pneumoniae surface associated PPP having a molecularweight of about 20 kDa, where the molecular weight is determined using a10–20% SDS-PAGE gel, or a fragment thereof, the PPP encoded by a nucleicacid sequence having a sequence as depicted in SEQ ID NO: 4, or animmunogenic fragment thereof; (ii) a pharmaceutically acceptablecarrier; and (iii) optionally at least one adjuvant.

The present invention also contemplates an immunogenic compositioncomprising (i) a S. pneumoniae surface associated PPP having a molecularweight of about 20 kDa, where the molecular weight is determined using a10–20% SDS-PAGE gel, or a fragment thereof; (ii) a pharmaceuticallyacceptable carrier; and (iii) optionally at least one adjuvant; wherethe composition elicits protective immunity from a disease caused byStreptococcus pneumoniae.

The present invention also contemplates an immunogenic compositioncomprising (i) a S. pneumoniae surface associated PPP having a molecularweight of about 20 kDa, where the molecular weight is determined using a10–20% SDS-PAGE gel, or a fragment thereof; (ii) a pharmaceuticallyacceptable carrier; and (iii) optionally at least one adjuvant; wherethe composition elicits protective immunity from a disease caused byStreptococcus pneumoniae; where the disease is selected from the groupconsisting of otitis media, rhinosinusitis, bacteremia, meningitis,pneumonia, and lower respiratory tract infection.

The present invention also contemplates an immunogenic compositioncomprising (i) a S. pneumoniae surface associated PPP having a molecularweight of about 20 kDa, where the molecular weight is determined using a10–20% SDS-PAGE gel, or a fragment thereof; (ii) a pharmaceuticallyacceptable carrier; and (iii) optionally at least one adjuvant; wherethe composition elicits protective immunity from a disease caused byStreptococcus pneumoniae; where the PPP comprises an amino acid sequenceas depicted in SEQ ID NO: 5, or an immunogenic fragment thereof.

The present invention also contemplates an immunogenic compositioncomprising (i) a S. pneumoniae surface associated PPP having a molecularweight of about 20 kDa, where the molecular weight is determined using a10–20% SDS-PAGE gel, or a fragment thereof where the PPP is encoded by anucleic acid sequence as depicted in SEQ ID NO: 4, or an immunogenicfragment thereof; (ii) a pharmaceutically acceptable carrier; and (iii)optionally at least one adjuvant; where the composition elicitsprotective immunity from a disease caused by Streptococcus pneumoniae.

The present invention contemplates an immunogenic composition comprising(i) at least one expression vector encoding a PPP having a molecularweight of about 20 kDa, where the molecular weight is determined using a10–20% SDS-PAGE gel; (ii) a pharmaceutically acceptable carrier; and(iii) optionally at least one adjuvant.

The present invention contemplates an immunogenic composition comprising(i) at least one expression vector encoding a PPP having a molecularweight of about 20 kDa, where the molecular weight is determined using a10–20% SDS-PAGE gel; (ii) a pharmaceutically acceptable carrier; and(iii) optionally at least one adjuvant; where the pneumococcal bacteriais Streptococcus pneumoniae.

The present invention contemplates an immunogenic composition comprising(i) at least one expression vector encoding a PPP having a molecularweight of about 20 kDa, where the molecular weight is determined using a10–20% SDS-PAGE gel; (ii) a pharmaceutically acceptable carrier; and(iii) optionally at least one adjuvant; where the composition elicitsprotective immunity from a disease caused by Streptococcus pneumoniae.

The present invention contemplates an immunogenic composition comprising(i) at least one expression vector encoding a PPP having a molecularweight of about 20 kDa, where the molecular weight is determined using a10–20% SDS-PAGE gel; (ii) a pharmaceutically acceptable carrier; and(iii) optionally at least one adjuvant; where the composition elicitsprotective immunity from a disease caused by Streptococcus pneumoniae;where the disease is selected from the group consisting of otitis media,rhinosinusitis, bacterenia, meningitis, pneumonia, and lower respiratorytract infection.

The present invention contemplates an immunogenic composition comprising(i) at least one expression vector encoding a PPP having a molecularweight of about 20 kDa, where the molecular weight is determined using a10–20% SDS-PAGE gel, where the PPP has an isoelectric point of about4.587; (ii) a pharmaceutically acceptable carrier; and (iii) optionallyat least one adjuvant.

The present invention contemplates an immunogenic composition comprising(i) at least one expression vector encoding a PPP having a molecularweight of about 20 kDa, where the molecular weight is determined using a10–20% SDS-PAGE gel, where the PPP has an isoelectric point of about4.587 and has a charge of about 14.214 at pH7; (ii) a pharmaceuticallyacceptable carrier; and (iii) optionally at least one adjuvant.

The present invention contemplates an immunogenic composition comprising(i) at least one expression vector encoding a PPP having a molecularweight of about 20 kDa, where the molecular weight is determined using a10–20% SDS-PAGE gel where expression vector comprises a nucleic acidsequence encoding an amino acid sequence as depicted in SEQ ID NO: 5, oran immunogenic fragment thereof; (ii) a pharmaceutically acceptablecarrier; and (iii) optionally at least one adjuvant.

The present invention contemplates an immunogenic composition comprising(i) at least one expression vector encoding a PPP having a molecularweight of about 20 kDa, where the molecular weight is determined using a10–20% SDS-PAGE gel where the expression vector comprises a nucleic acidsequence encoding an amino acid sequence as depicted in SEQ ID NO: 5, oran immunogenic fragment thereof; (ii) a pharmaceutically acceptablecarrier; and (iii) optionally at least one adjuvant.

The present invention contemplates an immunogenic composition comprising(i) at least one expression vector encoding a PPP having a molecularweight of about 20 kDa, where the molecular weight is determined using a10–20% SDS-PAGE gel where the expression vector comprises a nucleic acidsequence depicted in SEQ ID NO:4, or an immunogenic fragment thereof;(ii) a pharmaceutically acceptable carrier; and (iii) optionally atleast one adjuvant.

The present invention contemplates a method of inducing an immuneresponse in a mammal, the method comprising administering to the mammalan amount of a composition effective to induce an immune response in themammal; where the composition comprises (i) a S. pneumoniae surfaceassociated PPP having a molecular weight of about 20 kilo Daltons (kDa),wherein said molecular weight is determined using a 10–20% SDS-PAGE gel,or a fragment thereof; (ii) a pharmaceutically acceptable carrier; and(iii) optionally at least one adjuvant.

The present invention contemplates a method of inducing an immuneresponse in a mammal, the method comprising administering to the mammalan amount of an immunogenic composition effective to induce an immuneresponse in the mammal; where the composition comprises (i) a S.pneumoniae surface associated PPP having a molecular weight of about 20kDa, where the molecular weight is determined using a 10–20% SDS-PAGEgel, or a fragment thereof, which PPP has an amino acid sequence asdepicted in SEQ ID NO: 5, or an immunogenic fragment thereof; (ii) apharmaceutically acceptable carrier; and (iii) optionally at least oneadjuvant.

The present invention contemplates a method of inducing an immuneresponse in a mammal, the method comprising administering to the mammalan amount of an immunogenic composition effective to induce an immuneresponse in the mammal; where the composition comprises (i) at least oneexpression vector encoding a PPP having a molecular weight of about 20kDa, wherein said molecular weight is determined using a 10–20% SDS-PAGEgel, where the PPP having an isoelectric point of about 4.582; (ii) apharmaceutically acceptable carrier; and (iii) optionally at least oneadjuvant.

The present invention contemplates a method of inducing an immuneresponse in a mammal, the method comprising administering to the mammalan amount of a composition effective to induce an immune response in themammal; where the composition comprises (i) at least one expressionvector encoding a PPP having a molecular weight of about 20 kDa, whereinsaid molecular weight is determined using a 10–20% SDS-PAGE gel; (ii) apharmaceutically acceptable carrier; and (iii) optionally at least oneadjuvant; wherein said expression vector comprises a nucleic acidsequence encoding an amino acid sequence as depicted in SEQ ID NO: 5, oran immunogenic fragment thereof.

The present invention contemplates a method of inducing an immuneresponse in a mammal which is infected with pneumococcal bacteria, themethod comprising administering to the mammal an amount of a compoundeffective to inhibit binding of an amino acid sequence as depicted inSEQ ID NO: 5 to induce the immune response in the mammal.

The present invention also contemplates a method for screening for acompound which induces an immune response in a mammal infected withpneumococcal bacteria, the method comprising comparing a first amount ofbinding of an amino acid sequence as depicted in SEQ ID NO: 5 in thepresence of the compound to a second amount of binding of an amino acidsequence as depicted in SEQ ID NO: 5 not in the presence of thecompound; whereby a lower first amount of binding than the second amountbinding indicates that the compound may induce the immune response inthe mammal.

The present invention also contemplates a method for diagnosingpneumococcal bacterial infection, the method comprising comparing thelevel of PPP as depicted in SEQ ID NO: 5, or fragments thereof, insuspect sample to the level of PPP as depicted in SEQ ID NO: 5, orfragments thereof, in a control sample, whereby a higher level of thePneumo Protective Protein the suspect sample than the level of thePneumo Protective Protein in the control sample indicates that thesuspect sample comprises pneumococcal bacterial infection.

The present invention further contemplates an antibody which binds toStreptococcus pneumoniae PPP.

The present invention also contemplates an antibody which binds toStreptococcus pneumoniae PPP, which selectively recognizes an amino acidsequence as depicted in SEQ ID NO: 5, or fragments thereof.

The present invention also contemplates a chimeric antibody which bindsto Streptococcus pneumoniae PPP.

The present invention also contemplates a humanized antibody which bindsto Streptococcus pneumoniae PPP.

The present invention also contemplates an anti-idiotypic antibody whichbinds to Streptococcus pneumoniae PPP.

The present invention also contemplates an antibody which binds toStreptococcus pneumoniae PPP, where the antibody is conjugated to apharmaceutically active compound.

The present invention also contemplates a monoclonal antibody whichbinds to Streptococcus pneumoniae PPP.

The present invention also contemplates a monoclonal antibody whichbinds to Streptococcus pneumoniae PPP, where the antibody is humanized.

The present invention also contemplates a monoclonal antibody whichbinds to Streptococcus pneumoniae PPP, where the antibody isanti-idiotypic.

The present invention also contemplates a monoclonal antibody whichbinds to Streptococcus pneumoniae PPP, where the antibody is conjugatedto a pharmaceutically active compound.

The present invention contemplates a method for inducing an immuneresponse in a mammal, the method comprising administering to the mammalan amount of an anti-idiotypic antibody which binds to Streptococcuspneumoniae PPP which is effective to induce an immune response in themammal.

The present invention contemplates a method for inducing an immuneresponse in a mammal, the method comprising administering to the mammalan amount of a monoclonal antibody which binds to Streptococcuspneumoniae PPP, where the antibody is anti-idiotypic; effective toinduce an immune response in the mammal.

The present invention contemplates a method for inducing an immuneresponse in a mammal infected with pneumococcal bacteria, the methodcomprising administering to the mammal an amount of an antibody whichbinds to Streptococcus pneumoniae PPP, where the antibody is conjugatedto a pharmaceutically active compound; effective to induce an immuneresponse in the mammal.

The present invention also contemplates a method for inducing an immuneresponse in a mammal infected with pneumococcal bacteria, the methodcomprising administering to the mammal an amount of a monoclonalantibody which binds to Streptococcus pneumoniae PPP, where the antibodyis conjugated to a pharmaceutically active compound; effective to inducean immune response in the mammal.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. SDS-PAGE gel of DEAE fractions from PBS washes of S. pneumoniaestrain 49136. Lane 1 is unstained standards; lane 2 is fraction #8; lane3 is fraction #9; lane 4 is fraction #10; lane 5 is fraction #11; lane 6is fraction #12; lane 7 is fraction #13; lane 8 is fraction #19; lane 9is fraction #15; and lane 10 is fraction #16. The gel in FIG. 1 showsthe distinct small molecular weight band in fractions #14 and #15 (lanes8 and 9) resolved by the gel.

FIG. 2. Gel of whole cell lysate of recombinant expression of pLP533showing expression of the desired product. Lane 1, Biorad prestainedmarkers; Lane 2, uninduced cells; Lane 3, induced cells.

FIG. 3. Western blot of whole cell lysates of several serotypes showingcross reactivity and oligomer formation. Lane 1, Biorad Precisionprestained markers; lane 2, type 3; lane 3, type 4; lane 4, type 9; lane5, type 14; lane 6, type 19F; lane 7, type 18C; lane 8, type 5; and lane9, tupe 23F.

FIG. 4. Reduction of colonization by rPPP1. Bacteria recovered shown asLog10 CFU/gram of tissue. One standard error of the mean is shown.*values are significantly different compared to the control byTukey-Kramer statistical test.

FIG. 5. SDS-PAGE gel shows purification of rPPP1. Lane 1, Bio RadPrecision standards; lane 2, diafiltrate; lane 3, purified rPPP1.

FIGS. 6A and 6B. Comparison of sequences of PPP1 from serotypes of S.pneumoniae.

FIG. 7. Gel shows amplified PPP1 from in vitro and in vivo cultures.

DETAILED DESCRIPTION

The proteins and nucleic acids of this invention possess diagnostic,prophylactic and therapeutic utility for diseases caused byStreptococcus pneumoniae infection. They can be used to design screeningsystems for compounds that interfere or disrupt interaction of proteinsassociated with S. pneumoniae with iron. The nucleic acids and proteinsalso can be used in the preparation of compositions against S.pneumoniae infection and/or other pathogens when used to express foreigngenes.

In the present invention, a recombinant 20 kDa protein from whole S.pneumoniae that reduces colonization of S. pneumoniae, in an intranasalchallenge model, has been identified. The protein described herein hasbeen named Pneumo Protective Protein 1 (PPP1 ). This protein showssignificant homology to a non heme containing ferretin protein from L.innocua, which interestingly, is a member of the Dps family of DNAbinding proteins (Pikis, A., et al., J. Infect. Diseases, 1998,178:700). The ability of this protein to reduce colonization was thusunexpected, due to its predicted location in the cytoplasm.

Chemical studies indicate that the isolated S. pneumoniae surfaceassociated PPP has a molecular weight of about 20 kDa, where themolecular weight is determined using a 10–20% SDS-PAGE gel. Therecombinant PPP is determined to have an isoelectric point of about4.587. Additionally, the protein has a charge of about −14.214 at pH ofabout 7.

Streptococcus Pneumoniae

S. pneumoniae is a species of bacteria which is highly infectious in thehuman body. There have been more than 80 serotypes identified, to date.Several of these serotypes are etiological agents in a variety ofdisease states including, but not limited to, pneumonia, meningitis,endocarditis, arthritis, sinusitis, otitis, bronchitis, and laryngitis.Pneumococcal infections have been identified as a leading cause of deathin persons with immunocompromised systems, such as those infected withHIV.

S. pneumoniae is a species of the Streptococcus genus of theStreptococceaceae family. This family comprises Gram-positive,non-motile, spherical or oval cells that do not form endospores. S.pneumoniae have an inorganic terminal electron acceptor foroxidative-metabolism; however, they will grow in the presence of oxygen.This allows S. pneumoniae to grow in a variety of environments and thusit is well adapted to grow in various human tissues. The bacteria isdifficult to target with penicillin, since many strains produce apolysaccharide capsule.

The first step towards pneumococcal infection is colonization of thenasopharynx. Disruption of binding of the pneumococci to humannasopharyngeal/otic cells should result in reduction of colonization anda lower disease potential. Thus, isolation of the structures responsiblefor pneumococcal binding to human cells could lead to vaccinecandidates. Pneumococci have evolved numerous mechanisms for binding tohuman nasopharyngeal cells, including the PspA, PsaA, and CbpA proteins.Additionally, pneumococci may specifically bind to human nasopharyngealmucin as a first step in colonization. Thus, identification of thepneumococcal structure(s) responsible for this interaction may identifypotential vaccine targets.

Molecular Biology

Embodiments of this invention relate to isolated polynucleotidesequences encoding the polypeptides or proteins, as well as variants ofsuch sequences. Preferably, under high stringency conditions, thesevariant sequences hybridize to polynucleotides encoding one or morepneumo protective proteins. More preferably, under high stringencyconditions, these variant sequences hybridize to polynucleotidesencoding one or more pneumo protective protein sequences, such as thepolynucleotide sequence of SEQ ID NO: 4. For the purposes of defininghigh stringency southern hybridization conditions, reference canconveniently be made to Sambrook et al. (1989) at pp. 387–389 which isherein incorporated by reference, where the washing step is consideredhigh stringency.

This invention also relates to conservative variants wherein thepolynucleotide sequence differs from a reference sequence through achange to the third nucleotide of a nucleotide triplet. Preferably theseconservative variants function as biological equivalents to the PPP1reference polynucleotide sequence. In a preferred embodiment, variantsthat function as biological equivalents are those that bind to iron.

The present invention further comprises DNA sequences which, by virtueof the redundancy of the genetic code, are biologically equivalent tothe sequences which encode for the PPP1, that is, these other DNAsequences are characterized by nucleotide sequences which differ fromthose set forth herein, but which encode a protein having the same aminoacid sequence as that encoded by the DNA sequence in SEQ ID NO: 4.

This invention also comprises DNA sequences which encode amino acidsequences which differ from those of the S. pneumonia PPP1, but whichare biologically equivalent to those described for this protein (SEQ IDNO: 5). Such amino acid sequences may be said to be biologicallyequivalent to such PPP1 if their sequences differ only by minordeletions from, insertions into or substitutions to the PPP1 sequence,such that the tertiary configurations of the sequences are essentiallyunchanged from those of the wild-type protein.

For example, a codon for the amino acid alanine, a hydrophobic aminoacid, may be substituted by a codon encoding another less hydrophobicresidue, such as glycine, or a more hydrophobic residue, such as valine,leucine, or isoleucine. Similarly, changes which result in substitutionof one negatively charged residue for another, such as aspartic acid forglutamic acid, or one positively charged residue for another, such aslysine for arginine, as well as changes based on similarities ofresidues in their hydropathic index, can also be expected to produce abiologically equivalent product. Nucleotide changes which result inalteration of the N-terminal or C-terminal portions of the proteinmolecule would also not be expected to alter the activity of theprotein.

One can use the hydropathic index of amino acids in conferringinteractive biological function on a polypeptide, as discussed by Kyteand Doolittle (1982), wherein it was determined that certain amino acidsmay be substituted for other amino acids having similar hydropathicindices and still retain a similar biological activity. Alternatively,substitution of like amino acids may be made on the basis ofhydrophilicity, particularly where the biological function desired inthe polypeptide to be generated is intended for use in immunologicalembodiments. See, for example, U.S. Pat. No. 4,554,101 (which is herebyincorporated herein by reference), which states that the greatest localaverage hydrophilicity of a “protein,” as governed by the hydrophilicityof its adjacent amino acids, correlates with its immunogenicity.Accordingly, it is noted that substitutions can be made based on thehydrophilicity assigned to each amino acid. In using either thehydrophilicity index or hydropathic index, which assigns values to eachamino acid, it is preferred to introduce substitutions of amino acidswhere these values are±2, with±1 being particularly preferred, and thosewithin±0.5 being the most preferred substitutions.

Furthermore, changes in known variable regions are biologicallyequivalent where the tertiary configurations of the conserved regionsare essentially unchanged from those of PPP1. An alternative definitionof a biologically equivalent sequence is one that is still capable ofgenerating a cross-reactive immune response. In particular, the proteinsmay be modified by lengthening or shortening the corresponding insertionfrom the gonococcal pilin, as long as the modified protein is stillcapable of generating a desired immune response.

Each of the proposed modifications is well within the routine skill inthe art, as is determination of retention of structural and biologicalactivity of the encoded products. Therefore, where the terms “pneumoprotective protein”, or “PPP1”, or “PPP” are used in either thespecification or the claims, it will be understood to encompass all suchmodifications and variations which result in the production of abiologically equivalent protein.

Preferable characteristics of PPP1 described herein, encoded by thenucleotide sequences of this invention, include one or more of thefollowing: (a) being a membrane protein or being a protein directlyassociated with a membrane; (b) capable of being separated as a proteinusing an SDS acrylamide gel; and (c) retaining its biological functionof interacting with iron.

Variants and fragments may be attenuated, i.e. having reduced on noiron-binding activity when compared to wild-type PPP1 of the presentinvention. Preferably, the fragments and variant amino acid sequencesand variant nucleotide sequences expressing PPP1 are biologicalequivalents, i.e. they retain substantially the same function of thewild-type PPP1. Such variant amino acid sequences are encoded bypolynucleotides sequences of this invention. Such variant amino acidsequences may have about 70% to about 80%, and preferably about 90%,overall similarity to the amino acid sequence of PPP1. In a preferredembodiment, these sequences are shown in FIG. 6 and SEQ ID NOs10–19. Thevariant nucleotide sequences may have either about 70% to about 80%, andpreferably about 90%, overall similarity to the nucleotide sequenceswhich, when transcribed, encode the amino acid sequence of PPP1 or avariant amino acid sequence of PPP1. The attenuated proteins of thepresent invention comprise at least one epitopic region of the wild-typeprotein. In alternative embodiments, the epitopic region of the proteincomprises at least 20 contiguous nucleotides or 8 contiguous aminoacids.

The invention further relates to the overall consensus sequence of PPP1.Deduced amino acid sequences of PPP1 from different serotypes of S.pneumoniae may be compared to determine the conserved sequences. In aone embodiment, 10 different serotypes are compared. The conservedsequence may have many uses such as, but not limited to, determining theminimal requirements needed for protein binding, activity, and/orfunction. In a preferred embodiment, the consensus sequence of PPP1 isdepicted in FIG. 6 and SEQ ID NO:20.

The “isolated” sequences of the present invention are non-naturallyoccurring sequences. For example, these sequences can be isolated fromtheir normal state within the genome of the bacteria; or the sequencesmay be synthetic, i.e. generated via recombinant techniques, such aswell-known recombinant expression systems, or generated by a machine.

The invention also provides a recombinant DNA cloning vehicle capable ofexpressing a PPP1 comprising an expression control sequence havingpromoter and initiator sequences and a nucleic acid sequence of thepresent invention located 3′ to the promoter and initiator sequences.Cloning vehicles can be any plasmid or expression vector known in theart, including viral vectors (see below). In a further aspect, there isprovided a host cell containing a recombinant DNA cloning vehicle and/ora recombinant PPP1 of the present invention. Suitable expression controlsequences, host cells and expression vectors are well known in the art,and are described by way of example, in Sambrook et al. (1989).

Suitable host cells may be selected based on factors which can influencethe yield of recombinantly expressed proteins. These factors include,but are not limited to, growth and induction conditions, mRNA stability,codon usage, translational efficiency and the presence oftranscriptional terminators to minimize promoter read through. Uponselection of suitable host cells, the cell may be transfected withexpression vectors comprising nucleic acid sequences of the presentinvention. The cells may be transfected using any methods known in theart (see below).

Once host cells have been transfected with expression vectors of thepresent invention, cells are cultured under conditions such thatpolypeptides are expressed. The polypeptide is then isolatedsubstantially free of contaminating host cell components by techniquesthat are well known to those skilled in the art.

Depending on the application of the desired recombinant proteins, aheterologous nucleotide sequence may encode a co-factor, cytokine (suchas an interleukin), a T-helper epitope, a restriction marker, adjuvant,or a protein of a different microbial pathogen (e.g. virus, bacterium,fungus or parasite), especially proteins capable of eliciting aprotective immune response. It may be desirable to select a heterologoussequence that encodes an immunogenic portion of a co-factor, cytokine(such as an interleukin), a T-helper epitope, a restriction marker,adjuvant, or a protein of a different microbial pathogen (e.g. virus,bacterium or fungus). Other types of non-PPP1 moieties include, but arenot limited to, those from cancer cells or tumor cells, allergens,amyloid peptide, protein or other macromolecular components.

For example, in certain embodiments, the heterologous genes encodecytokines, such as interleukin-12, which are selected to improve theprophylatic or therapeutic characteristics of the recombinant proteins.

Examples of such cancer cells or tumor cells include, but are notlimited to, prostate specific antigen, carcino-embryonic antigen, MUC-1,Her2, CA-125 and MAGE-3.

Examples of such allergens include, but are not limited to, thosedescribed in U.S. Pat. No. 5,830,877 and published International PatentApplication Number WO 99/51259, which are hereby incorporated byreference, and include pollen, insect venoms, animal dander, fungalspores and drugs (such as penicillin). Such components interfere withthe production of IgE antibodies, a known cause of allergic reactions.

Amyloid peptide protein (APP) has been implicated in diseases referredto variously as Alzheimer's disease, amyloidosis or amyloidogenicdisease. The β-amyloid peptide (also referred to as Aβ peptide) is a 42amino acid fragment of APP, which is generated by processing of APP bythe β and γ secretase enzymes, and has the following sequence:

Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys Leu ValPhe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile Gly Leu Met ValGly Gly Val Val Ile Ala (SEQ ID NO: 6).

In some patients, the amyloid deposit takes the form of an aggregated Aβpeptide. Surprisingly, it has now been found that administration ofisolated Aβ peptide induces an immune response against the Aβ peptidecomponent of an amyloid deposit in a vertebrate host (See PublishedInternational Patent Application WO 99/27944). Such Aβ peptides havealso been linked to unrelated moieties. Thus, the heterologousnucleotides sequences of this invention include the expression of thisAβ peptide, as well as fragments of Aβ peptide and antibodies to Aβpeptide or fragments thereof. One such fragment of Aβ peptide is the 28amino acid peptide having the following sequence (as disclosed in U.S.Pat. No. 4,666,829):

Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys Leu ValPhe Phe Ala Glu Asp Val Gly Ser Asn Lys (SEQ ID NO: 7).

The heterologous nucleotide sequence can be selected to make use of thenormal route of infection of pneumococcal bacteria, which enters thebody through the respiratory tract and can infect a variety of tissuesand cells, for example, the meninges, blood, and lung. The heterologousgene may also be used to provide agents which are used for gene therapyor for the targeting of specific cells. As an alternative to merelytaking advantage of the normal cells exposed during the normal route ofpneumococcal infection, the heterologous gene, or fragment, may encodeanother protein or amino acid sequence from a different pathogen which,when employed as part of the recombinant protein, directs therecombinant protein to cells or tissue which are not in the normal routeof infection. In this manner, the protein becomes a targeting tool forthe delivery of a wider variety of foreign proteins.

Molecular weight of proteins may be determined by using any method knownin the art. A non-limiting list of methods includes, denaturing SDS-PAGEgel, size exclusion chromatography, and iso-electric focusing.Conditions appropriate for each method (e.g. time of separation,voltage, current, and buffers) can be determined as needed using definedmethods in the art. In a preferred embodiment, denaturing SDS-PAGE isused to determine the molecular weight of the proteins. Additionally,the conditions used to determine the molecular weight are preferably, 1hour separation time at 20 milli Amps and constant current.

Detection of the proteins can be determined using various methods in theart. These methods include, but are not limited to, Western blotting,coomassie blue staining, silver staining, autoradiography, fluorescentand phosphorescent probing. In a preferred embodiment of this invention,the proteins were detected by Western blotting.

The terms “pneumo protective protein”, “PPP1”, and “PPP” in describingembodiments of the invention, infra, includes embodiments that employfragments, variants and attenuated forms thereof as a replacement forwild-type PPP1 or as addition thereto, unless specified otherwise.

Viral and Non-Viral Vectors

Preferred vectors, particularly for cellular assays in vitro and invivo, are viral vectors, such as lentiviruses, retroviruses, herpesviruses, adenoviruses, adeno-associated viruses, vaccinia virus,baculovirus, alphaviruses and other recombinant viruses with desirablecellular tropism. Thus, a gene encoding a functional or mutant proteinor polypeptide domain fragment thereof can be introduced in vivo, exvivo, or in vitro using a viral vector or through direct introduction ofDNA. Expression in targeted tissues can be effected by targeting thetransgenic vector to specific cells, such as with a viral vector or areceptor ligand, or by using a tissue-specific promoter, or both.Targeted gene delivery is described in PCT Publication No. WO 95/28494.

Viral vectors commonly used for in vivo or ex vivo targeting and therapyprocedures are DNA-based vectors and retroviral vectors. Methods forconstructing and using viral vectors are known in the art (e.g., Millerand Rosman, BioTechniques, 1992, 7:980–990). Preferably, the viralvectors are replication-defective, that is, they are unable to replicateautonomously in the target cell. Preferably, the replication defectivevirus is a minimal virus, i.e., it retains only the sequences of itsgenome which are necessary for encapsulating the genome to produce viralparticles.

Examples of alphaviruses include, but are not limited to, Eastern EquineEncephalitis virus (EEE), Venezuelan Equine Encephalitis virus (VEE),Everglades virus, Mucambo virus, Pixuna virus, Western EquineEncephalitis virus (WEE), Sindbis virus, Semliki Forest virus,Middelburg virus, Chikungunya virus, O'nyong-nyong virus, Ross Rivervirus, Barmah Forest virus, Getah virus, Sagiyama virus, Bebaru virus,Mayaro virus, Una virus, Aura virus, Whataroa virus, Babanki virus,Kyzylagach virus, Highlands J virus, Fort Morgan virus, Ndumu virus, andBuggy Creek virus (U.S. Pat. No. 6,156,558).

DNA viral vectors include an attenuated or defective DNA virus, such asbut not limited to herpes simplex virus (HSV), papillomavirus, EpsteinBarr virus (EBV), adenovirus, adeno-associated virus (AAV), and thelike. Defective viruses, which entirely or almost entirely lack viralgenes, are preferred. Defective virus is not infective afterintroduction into a cell. Use of defective viral vectors allows foradministration to cells in a specific, localized area, without concernthat the vector can infect other cells. Thus, a specific tissue can bespecifically targeted. Examples of particular vectors include, but arenot limited to, a defective herpes virus 1 (HSV1) vector (Kaplitt etal., Molec. Cell. Neurosci., 1991, 2:320–330), defective herpes virusvector lacking a glyco-protein L gene, or other defective herpes virusvectors (PCT Publication Nos. WO 94/21807 and WO 92/05263); anattenuated adenovirus vector, such as the vector described byStratford-Perricaudet et al. (J. Clin. Invest., 1992, 90:626–630; seealso La Salle et al., Science, 1993, 259:988–990); and a defectiveadeno-associated virus vector (Samulski et al., J. Virol., 1987,61:3096–3101; Samulski et al., J. Virol., 1989, 63:3822–3828; Lebkowskiet al., Mol. Cell. Biol., 1988, 8:3988–3996).

Various companies produce viral vectors commercially, including, but notlimited to, Avigen, Inc. (Alameda, Calif.; AAV vectors), Cell Genesys(Foster City, Calif.; retroviral, adenoviral, AAV vectors, andlentiviral vectors), Clontech (retroviral and baculoviral vectors),Genovo, Inc. (Sharon Hill, Pa.; adenoviral and AAV vectors), Genvec(adenoviral vectors), IntroGene (Leiden, Netherlands; adenoviralvectors), Molecular Medicine (retroviral, adenoviral, AAV, and herpesviral vectors), Norgen (adenoviral vectors), Oxford BioMedica (Oxford,United Kingdom; lentiviral vectors), and Transgene (Strasbourg, France;adenoviral, vaccinia, retroviral, and lentiviral vectors).

Adenovirus vectors. Adenoviruses are eukaryotic DNA viruses that can bemodified to efficiently deliver a nucleic acid of the invention to avariety of cell types. Various serotypes of adenovirus exist. Of theseserotypes, preference is given, within the scope of the presentinvention, to using type 2 or type 5 human adenoviruses (Ad 2 or Ad 5)or adenoviruses of animal origin (see PCT Publication No. WO 94/26914).Those adenoviruses of animal origin which can be used within the scopeof the present invention include adenoviruses of canine, bovine, murine(example: Mav1, Beard et al., Virology, 1990, 75–81), ovine, porcine,avian, and simian (example: SAV) origin. Preferably, the adenovirus ofanimal origin is a canine adenovirus, more preferably a CAV2 adenovirus(e.g., Manhattan or A26/61 strain, ATCC VR-800, for example). Variousreplication defective adenovirus and minimum adenovirus vectors havebeen described (PCT Publication Nos. WO 94/26914, WO 95/02697, WO94/28938, WO 94/28152, WO 94/12649, WO 95/02697, WO 96/22378). Thereplication defective recombinant adenoviruses according to theinvention can be prepared by any technique known to the person skilledin the art (Levrero et al., Gene, 1991, 101:195; European PublicationNo. EP 185 573; Graham, EMBO J., 1984, 3:2917; Graham et al., J. Gen.Virol., 1977, 36:59). Recombinant adenoviruses are recovered andpurified using standard molecular biological techniques, which are wellknown to one of ordinary skill in the art.

Adeno-associated viruses. The adeno-associated viruses (AAV) are DNAviruses of relatively small size that can integrate, in a stable andsite-specific manner, into the genome of the cells which they infect.They are able to infect a wide spectrum of cells without inducing anyeffects on cellular growth, morphology or differentiation, and they donot appear to be involved in human pathologies. The AAV genome has beencloned, sequenced and characterized. The use of vectors derived from theAAVs for transferring genes in vitro and in vivo has been described(see, PCT Publication Nos. WO 91/18088 and WO 93/09239; U.S. Pat. Nos.4,797,368 and 5,139,941; European Publication No. EP 488 528). Thereplication defective recombinant AAVs according to the invention can beprepared by cotransfecting a plasmid containing the nucleic acidsequence of interest flanked by two AAV inverted terminal repeat (ITR)regions, and a plasmid carrying the AAV encapsidation genes (rep and capgenes), into a cell line which is infected with a human helper virus(for example an adenovirus). The AAV recombinants which are produced arethen purified by standard techniques.

Retrovirus vectors. In another embodiment the gene can be introduced ina retroviral vector, e.g., as described in U.S. Pat. No. 5,399,346; Mannet al, Cell, 1983, 33:153; U.S. Pat. Nos. 4,650,764 and 4,980,289;Markowitz et al., J. Virol., 1988, 62:1120; U.S. Pat. No. 5,124,263;European Publication Nos. EP 453 242 and EP178 220; Bernstein et al.,Genet. Eng.,1985,7:235; McCormick, BioTechnology, 1985, 3:689; PCTPublication No. WO 95/07358; and Kuo et at., Blood, 1993, 82:845. Theretroviruses are integrating viruses that infect dividing cells. Theretrovirus genome includes two LTRs, an encapsidation sequence and threecoding regions (gag, pol and env). In recombinant retroviral vectors,the gag, pol and env genes are generally deleted, in whole or in part,and replaced with a heterologous nucleic acid sequence of interest.These vectors can be constructed from different types of retrovirus,such as, HIV, MoMuLV (“murine Moloney leukaemia virus” MSV (“murineMoloney sarcoma virus”), HaSV (“Harvey sarcoma virus”); SNV (“spleennecrosis virus”); RSV (“Rous sarcoma virus”) and Friend virus. Suitablepackaging cell lines have been described in the prior art, in particularthe cell line PA317 (U.S. Pat. No. 4,861,719); the PsiCRIP cell line(PCT Publication No. WO 90/02806) and the GP+envAm−12 cell line (PCTPublication No. WO 89/07150). In addition, the recombinant retroviralvectors can contain modifications within the LTRs for suppressingtranscriptional activity as well as extensive encapsidation sequenceswhich may include a part of the gag gene (Bender et al., J. Virol.,1987, 61:1639). Recombinant retroviral vectors are purified by standardtechniques known to those having ordinary skill in the art.

Retroviral vectors can be constructed to function as infectiousparticles or to undergo a single round of transfection. In the formercase, the virus is modified to retain all of its genes except for thoseresponsible for oncogenic transformation properties, and to express theheterologous gene. Non-infectious viral vectors are manipulated todestroy the viral packaging signal, but retain the structural genesrequired to package the co-introduced virus engineered to contain theheterologous gene and the packaging signals. Thus, the viral particlesthat are produced are not capable of producing additional virus.

Retrovirus vectors can also be introduced by DNA viruses, which permitsone cycle of retroviral replication and amplifies tranfection efficiency(see PCT Publication Nos. WO 95/22617, WO 95/26411, WO 96/39036 and WO97/19182).

Lentivirus vectors. In another embodiment, lentiviral vectors can beused as agents for the direct delivery and sustained expression of atransgene in several tissue types, including brain, retina, muscle,liver and blood. The vectors can efficiently transduce dividing andnondividing cells in these tissues, and maintain long-term expression ofthe gene of interest. For a review, see, Naldini, Curr. Opin.Biotechnol., 1998, 9:457–63; see also Zufferey, et al., J. Virol., 1998,72:9873–80). Lentiviral packaging cell lines are available and knowngenerally in the art. They facilitate the production of high-titerlentivirus vectors for gene therapy. An example is atetracycline-inducible VSV-G pseudotyped lentivirus packaging cell linethat can generate virusparticles at titers greater than 106 IU/ml for atleast 3 to 4 days (Kafri, et al., J. Virol., 1999, 73: 576–584). Thevector produced by the inducible cell line can be concentrated as neededfor efficiently transducing non-dividing cells in vitro and in vivo.

Non-viral vectors. In another embodiment, the vector can be introducedin vivo by lipofection, as naked DNA, or with other transfectionfacilitating agents (peptides, polymers, etc.). Synthetic cationiclipids can be used to prepare liposomes for in vivo transfection of agene encoding a marker (Felgner, et. al., Proc. Natl. Acad. Sci. U.S.A.,1987, 84:7413–7417; Felgner and Ringold, Science, 1989, 337:387–388; seeMackey, et al., Proc. Natl. Acad. Sci. U.S.A., 1988, 85:8027–8031; Ulmeret al., Science, 1993, 259:1745–1748). Useful lipid compounds andcompositions for transfer of nucleic acids are described in PCT PatentPublication Nos. WO 95/18863 and WO 96/17823, and in U.S. Pat. No.5,459,127. Lipids may be chemically coupled to other molecules for thepurpose of targeting (see Mackey, et. al., supra). Targeted peptides,e.g., hormones or neurotransmitters, and proteins such as antibodies, ornon-peptide molecules could be coupled to liposomes chemically.

Other molecules are also useful for facilitating transfection of anucleic acid in vivo, such as a cationic oligopeptide (e.g., PCT PatentPublication No. WO 95/21931), peptides derived from DNA binding proteins(e.g., PCT Patent Publication No. WO 96/25508), or a cationic polymer(e.g., PCT Patent Publication No. WO 95/21931).

It is also possible to introduce the vector in vivo as a naked DNAplasmid. Naked DNA vectors for gene therapy can be introduced into thedesired host cells by methods known in the art, e.g., electroporation,microinjection, cell fusion, DEAE dextran, calcium phosphateprecipitation, use of a gene gun, or use of a DNA vector transporter(e.g., Wu et al., J. Biol. Chem., 1992, 267:963–967; Wu and Wu, J. Biol.Chem., 1988, 263:14621–14624; Canadian Patent Application No. 2,012,311;Williams et al., Proc. Natl. Acad. Sci. USA, 1991, 88:2726–2730).Receptor-mediated DNA delivery approaches can also be used (Curiel etal., Hum. Gene Ther., 1992, 3:147–154; Wu and Wu, J. Biol. Chem., 1987,262:4429–4432). U.S. Pat. Nos. 5,580,859 and 5,589,466 disclose deliveryof exogenous DNA sequences, free of transfection facilitating agents, ina mammal. Recently, a relatively low voltage, high efficiency in vivoDNA transfer technique, termed electrotransfer, has been described (Miret al., C. P. Acad. Sci., 1988, 321:893; PCT Publication Nos. WO99/01157; WO 99/01158; WO 99/01175).

Assay System

Any cell assay system that allows for assessing functional activities ofimmunogenic compositions and compounds that modulate binding of PPP1 toiron is contemplated by the present invention. In a specific embodiment,the assay can be used to identify compounds that interact with PPP1 todecrease binding of PPP1, described herein, to iron. This can beevaluated by assessing the effects of a test compound on the interactionthe protein described herein. A cell assay system that assesses theability of the compound to elicit opsonophagocytic antibodies against S.pneumoniae may also be utilized (Gray, B. M. 1990. Conjugate VaccinesSupplement p694–697).

Any convenient method that permits detection of the binding of iron withPPP are contemplated by the present invention. In a preferred embodimentof the invention, protein components of S. pneumoniae can be separatedon a polyacrylamide gel and transferred to a solid support. The supportthen may be probed with a labeled interacting component (e.g. iron). Thecomponent may be labeled with any label known in the art including, butnot limited to, radioactivity, enzyme-based, dye molecules, or aflourescent or phosphorescent tag. In a preferred embodiment, the labelis radioactive. The label may be detected by any means known in the art.For example, autoradiography, scintillation counter, or ultra-violetlight. In a preferred embodiment, the radiolabel is detected byautoradiography. Assays that amplify the signals from the probe are alsoknown, such as, for example, those that utilize biotin and avidin, andenzyme-labeled immunoassays, such as ELISA assays.

In Vitro Screening Methods

Candidate agents are added to assay systems, prepared by known methodsin the art, and the level of binding between iron and PPP1 is measured.Various in vitro systems can be used to analyze the effects of acompound on iron binding. Preferably, each experiment is performed morethan once, such as, for example, in triplicate at multiple differentdilutions of compound.

The screening system of the invention permits detection of bindinginhibitors. An inhibitor screen involves detecting interaction of ironand PPP1 when contacted with a compound that regulates interaction ofthese proteins. If a decrease in the binding of iron to PPP1 isdetected, then the compound is a candidate inhibitor. If no decrease isobserved, the compound does not alter the binding of iron to the proteinof the present invention.

Immunogenic Compositions

In further embodiments of this invention PPP1 are employed inimmunogenic compositions comprising (i) at least one PPP1; (ii) at leastone pharmaceutically acceptable buffer, diluent, or carrier; and (iii)optionally at least one adjuvant. In a preferred embodiment, theimmunogenic composition is used as a vaccine. The PPP1 may berecombinantly produced or isolated from a bacterial preparation,according to methods known in the art. Preferably, these compositionshave therapeutic and prophylactic applications as immunogeniccompositions in preventing, protecting and/or ameliorating pneumococcalinfection. In such applications, an immunologically effective amount ofat least one PPP1 is employed in such amount to cause a reduction,preferably a substantial reduction, in the course a normal pneumoccocalinfection. The proteins may be attenuated. The term “attenuated” refersto a protein that maintains its immunogenic activity, while one or moreother functional characteristics are decreased or deleted. For example,the attenuated form of this protein may exhibit diminished bindingproperties, such as its ability to bind iron. Alternatively, theattenuated form may decrease the ability of S. pneumoniae to bind iron.

As used herein, the term “effective amount” refers to amount of theimmunogen component (i.e. PPP1) described herein to stimulate an immuneresponse, i.e., to cause the production of antibodies and/or acell-mediated response when introduced into a subject. In a preferredembodiment, the effective amount will decrease the colonization of S.pneumoniae. The term “immunogen component” refers to the ability of thiscomponent to stimulate secretory antibody and/or cell-mediated responseproduction in local regions, e.g. nasopharynx, when administeredsystemically as an immunogenic composition according to the presentinvention.

As used herein the term “adjuvant” refers to an agent, compound or thelike, which potentiates or stimulates the immune response in a subjectwhen administered in combination with the immunogenic composition. Thus,the immune response, elicited by the immunogenic compositioncombination, as measured by any convention method known in the art, willgenerally be greater than that provoked by the immunogenic compositionalone.

The compositions of the invention can include an adjuvant, including,but not limited to aluminum hydroxide; aluminum phosphate; Stimulon™QS-21 (Aquila Biopharmaceuticals, Inc., Framingham, Mass.); MPL™(3-O-deacylated monophosphoryl lipid A; Corixa, Seattle, Wash.); RC529(Corixa) and aminoalkyl glucosamine phosphate compounds as described inPCT Published Application WO 98/50399 (RIBI Immunochem Research); IL-12(Genetics Institute, Cambridge, Mass.);N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP);N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to asnor-MDP);N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphos-phoryloxy)-ethylamine(CGP 19835A, referred to a MTP-PE); granulocyte-macrophage colonystimulating factor (GM-CSF) and cholera toxin. Others which may be usedare non-toxic derivatives of cholera toxin, including its B subunit (forexample, wherein glutamic acid at amino acid position 29 is replaced byanother amino acid, preferably, a histidine in accordance with PublishedInternational Patent Application WO 00/18434), and/or conjugates orgenetically engineered fusions of non-PPP polypeptides with choleratoxin or its B subunit, procholeragenoid, fungal polysaccharides. Theadjuvant may be used in its natural form or one can use a synthetic orsemi-synthetic version of an adjuvant. Any formulation of the adjuvantmay be used depending on the desired response and admininstrationmethod. Various forms of the adjuvant may be used, e.g., a liquid,powder or emulsion.

The immunogenic composition may be administered as a single bolus doseor as a “series” of administrations over a defined period of time (e.g.,one year). When given in later year, such series of administrations isreferred to as “booster shots”. These administrations increase theantibody levels produced by the previous administration. The immunogeniccompound may be administered until sufficient antibody levels have beenidentified in the subject, so as to induce an immune response uponchallenge from the immunogen.

The formulation of such immunogenic compositions is well known topersons skilled in this field. Immunogenic compositions of the inventionmay comprise additional antigenic components (e.g., polypeptide orfragment thereof or nucleic acid encoding an antigen or fragmentthereof) and, preferably, include a pharmaceutically acceptable carrier.Suitable pharmaceutically acceptable carriers and/or diluents includeany and all conventional solvents, dispersion media, fillers, solidcarriers, aqueous solutions, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like. The term“pharmaceutically acceptable carrier” refers to a carrier that does notcause an allergic reaction or other untoward effect in patients to whomit is administered. Suitable pharmaceutically acceptable carriersinclude, for example, one or more of water, saline, phosphate bufferedsaline, dextrose, glycerol, ethanol and the like, as well ascombinations thereof. Pharmaceutically acceptable carriers may furthercomprise minor amounts of auxiliary substances such as wetting oremulsifying agents, preservatives or buffers, which enhance the shelflife or effectiveness of the antigen. The use of such media and agentsfor pharmaceutically active substances is well known in the art. Exceptinsofar as any conventional media or agent is incompatible with theactive ingredient, use thereof in immunogenic compositions of thepresent invention is contemplated.

Compositions

In further embodiments of this invention, PPP1 nucleic acid sequences,amino acid sequences, expression vectors or host cells are employed incompositions comprising (i) at least one PPP1 protein, or nucleic acidencoding an amino acid sequence of a PPP1, or an expression vector orhost cell that expresses such nucleic acid arid (ii) at least one of apharmaceutically acceptable buffer, diluent, or carrier. The PPP1 may berecombinantly produced or isolated from a bacterial preparation,according to methods known in the art. Preferably, these compositionshave therapeutic and prophylactic applications. In such applications, apharmaceutically effective amount of at least one PPP1 is employed insuch amount to produce a defined functional activity. As used herein,the term “effective amount” refers to amount of the PPP1 proteindescribed herein, to produce a functional effect.

Administration of such compositons or immunogenic compositions may be byany conventional effective form, such as intranasally, parenterally,orally, or topically applied to mucosal surface such as intranasal,oral, eye, lung, vaginal, or rectal surface, such as by aerosol spray.The preferred means of administration is parenteral or intranasal.

Oral formulations include such normally employed excipients as, forexample, pharmaceutical grades of mannitol, lactose, starch, magnesiumstearate, sodium saccharine, cellulose, magnesium carbonate, and thelike.

The polynucleotides and polypeptides of the present invention may beadministered as the sole active immunogen in an immunogenic composition.Alternatively, however, the immunogenic composition may include otheractive immunogens, including other immunologically active antigens fromother pathogenic species. Preferably, the pathogenic species thatprovide other immunologically active antigens are bacterial pathogens,e.g., involved in bacterial infections. Indeed, preferably therapeuticuse of the PPP antigen of the invention will be as a component of amultivalent vaccine that includes other bacterial antigens from S.pneumonia or other pathogenic bacteria. The other immunologically activeantigens may be replicating agents or non-replicating agents.Replicating agents include, for example, attenuated forms of measlesvirus, rubella virus, variscella zoster virus (VZV), Parainfluenza virus(PIV), and Respiratory Syncytial virus (RSV).

One of the important aspects of this invention relates to a method ofinducing immune responses in a mammal comprising the step of providingto said mammal an immunogenic composition of this invention. Theimmunogenic composition is a composition which is immunogenic in thetreated animal or human such that the immunologically effective amountof the polypeptide(s) contained in such composition brings about thedesired response against pneumococcal infection. Preferred embodimentsrelate to a method for the treatment, including amelioration, orprevention of pneumococcal infection in a human comprising administeringto a human an immunologically effective amount of the immunogeniccomposition. The dosage amount can vary depending upon specificconditions of the individual. This amount can be determined in routinetrials by means known to those skilled in the art.

Certainly, the isolated amino acid sequences for the proteins of thepresent invention may be used in forming subunit immunogeniccompositions. They also may be used as antigens for raising polyclonalor monoclonal antibodies and in immunoassays for the detection ofanti-PPP1 protein-reactive antibodies. Immunoassays encompassed by thepresent invention include, but are not limited to, those described inU.S. Pat. No. 4,367,110. (double monoclonal antibody sandwich assay) andU.S. Pat. No. 4,452,901 (western blot), which U.S. Patents areincorporated herein by reference. Other assays includeimmunoprecipitation of labeled ligands and immunocytochemistry, both invitro and in vivo.

Methods of Inducing an Immune Response

According to the present invention, colonization of S. pneumoniaeinvolves PPP1 proteins. The present invention provides for methods thatprevent pneumococal infections by administering to a subject atherapeutically effective amount of an immunogenic composition thatinduces an immune response in the subject. These methods include, butare not limited to, administration of an immunogenic compositioncomprised of at least one PPP1 protein, variant, fragment or attenuatedversion thereof, or at least one expression vector encoding the proteinvariant, fragment or attenuated version thereof.

Methods of Inhibiting Pneumococcal Infection

The present invention further provides for methods to induce an immuneresponse in a subject which is infected with pneumococal bacteria byadministering to a subject a therapeutically effective amount of acomposition or compound that blocks functional effects associated withthe PPP1 proteins. These methods include, but are not limited to,administration of a composition comprised of at least one PPP1 proteinor fragments thereof or at least one expression vector encoding a PPP1protein or administration of a compound that blocks, substantially allor at least in part, a function of the PPP1 proteins.

Methods of Diagnosis

This invention also provides for a method of diagnosing a pneumococcalinfection, or identifying a pneumococcal immunogenic compositon strainthat has been administered, comprising the step of determining thepresence, in a sample, of an amino acid sequence of SEQ ID NO: 5 or anyof 10–19. Any conventional diagnostic method may be used. Thesediagnostic methods can easily be based on the presence of an amino acidsequence or polypeptide. Preferably, such a diagnostic method matchesfor a polypeptide having at least 10, and preferably at least 20, aminoacids which are common to the amino acid sequences of this invention.

The nucleic acid sequences disclosed herein also can be used for avariety of diagnostic applications. These nucleic acids sequences can beused to prepare relatively short DNA and RNA sequences that have theability to specifically hybridize to the nucleic acid sequences encodingthe PPP1 protein. Nucleic acid probes are selected for the desiredlength in view of the selected parameters of specificity of thediagnostic assay. The probes can be used in diagnostic assays fordetecting the presence of pathogenic organisms, or in identifying apneumococcal immunogenic composition that has been administered, in agiven sample. With current advanced technologies for recombinantexpression, nucleic acid sequences can be inserted into an expressionconstruct for the purpose of screening the corresponding oligopeptidesand polypeptides for reactivity with existing antibodies or for theability to generate diagnostic or therapeutic reagents. Suitableexpression control sequences and host cell/cloning vehicle combinationsare well known in the art, and are described by way of example, inSambrook et al. (1989).

In preferred embodiments, the nucleic acid sequences employed forhybridization studies or assays include sequences that are complementaryto a nucleotide stretch of at least about 10, preferably about 15, andmore preferably about 20 nucleotides. A variety of known hybridizationtechniques and systems can be employed for practice of the hybridizationaspects of this invention, including diagnostic assays such as thosedescribed in Falkow et al., U.S. Pat. No. 4,358,535. Preferably, thesequences recognize or bind a nucleic acid sequence on the PPP1 proteinare consecutive.

In general, it is envisioned that the hybridization probes describedherein will be useful both as reagents in solution hybridizations aswell as in embodiments employing a solid phase. In embodiments involvinga solid phase, the test DNA (or RNA) from suspected clinical samples,such as exudates, body fluids (e.g., middle ear effusion,bronchoalveolar lavage fluid) or even tissues, is absorbed or otherwiseaffixed to a selected matrix or surface. This fixed, single-strandednucleic acid is then subjected to specific hybridization with selectedprobes under desired conditions. The selected conditions will depend onthe particular circumstances based on the particular criteria required(depending, for example, on the G+C contents, type of target nucleicacid, source of nucleic acid, size of hybridization probe). Followingwashing of the hybridized surface so as to remove nonspecifically boundprobe molecules, specific hybridization is detected, or even quantified,by means of the label.

The nucleic acid sequences which encode the PPP1 protein of theinvention, or their variants, may be useful in conjunction with PCR*technology, as set out, e.g., in U.S. Pat. No. 4,603,102. One mayutilize various portions of any of the PPP1 protein sequences of thisinvention as oligonucleotide probes for the PCR* amplification of adefined portion of a PPP1 gene, or nucleotide, which sequence may thenbe detected by hybridization with a hybridization probe containing acomplementary sequence. In this manner, extremely small concentrationsof the PPP1 nucleic acid sequence may be detected in a sample utilizingthe nucleotide sequences of this invention.

The following examples are included to illustrate certain embodiments ofthe invention. However, those of skill in the art should, in the lightof the present disclosure, appreciate that many changes can be made inthe specific embodiments which are disclosed and still obtain a like orsimilar result without departing from the spirit and scope of theinvention.

Antibodies

The present invention describes antibodies that may be used to detectthe presence of PPP1 proteins present in samples. Additionally, theantibodies (e.g., anti-idiotypic antibodies) may be used to inhibitimmune responses to pneumococcal infections.

According to the invention, PPP1 protein polypeptides producedrecombinantly or by chemical synthesis, and fragments or otherderivatives, may be used as an immunogen to generate antibodies thatrecognize the polypeptide or portions thereof. The portion of thepolypeptide used as an immunogen may be specifically selected tomodulate immunogenicity of the developed antibody. Such antibodiesinclude, but are not limited to, polyclonal, monoclonal, humanized,chimeric, single chain, Fab fragments, and an Fab expression library. Anantibody that is specific for human PPP1 protein may recognize awild-type or mutant form of the PPP1 proteins. In a specific embodiment,the antibody is comprised of at least 8 amino acids, preferably from8–10 amino acids, and more preferably from 15–30 amino acids.Preferably, the antibody recognizes or binds amino acids on PPP1 areconsecutive.

Various procedures known in the art may be used for the production ofpolyclonal antibodies to polypeptides, derivatives, or analogs. For theproduction of antibody, various host animals, including but not limitedto rabbits, mice, rats, sheep, goats, etc, can be immunized by injectionwith the polypeptide or a derivative (e.g., fragment or fusion protein).The polypeptide or fragment thereof can be conjugated to an immunogeniccarrier, e.g., bovine serum albumin (BSA) or keyhole limpet hemocyanin(KLH). Various adjuvants may be used to increase the immunologicalresponse, depending on the host species, including but not limited toFreund's (complete and incomplete), mineral gels such as aluminumhydroxide, surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, KLH, dinitrophenol, andpotentially useful human adjuvants such as BCG ( bacilleCalmette-Guerin) and Corynebacterium parvum.

Monoclonal antibodies directed toward a PPP1 protein, fragment, analog,or derivative thereof, may be prepared by any technique that providesfor the production of antibody molecules by continuous cell lines inculture may be used. These include but are not limited to the hybridomatechnique originally developed by Kohler and Milstein (Nature256:495–497, 1975), as well as the trioma technique, the human B-cellhybridoma technique (Kozbor et al., Immunology Today 4:72, 1983; Cote etal., Proc. Natl. Acad. Sci. U.S.A. 80:2026–2030, 1983), and theEBV-hybridoma technique to produce human monoclonal antibodies (Cole etal., in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc.,pp. 77–96, 1985). “Chimeric antibodies” may be produced (Morrison etal., J. Bacteriol. 159:870, 1984; Neuberger et al., Nature 312:604–608,1984; Takeda et al., Nature 314:452–454, 1985) by splicing the genesfrom a non-human antibody molecule specific for a polypeptide togetherwith genes from a human antibody molecule of appropriate biologicalactivity.

In the production and use of antibodies, screening for or testing withthe desired antibody can be accomplished by techniques known in the art,e.g., radioimmunoassay, ELISA (enzyme-linked immunosorbant assay),“sandwich” immunoassays, immunoradiometric assays, gel diffusionprecipitin reactions, immunodiffusion assays, in situ immunoassays(using colloidal gold, enzyme or radioisotope labels, for example),western blots, precipitation reactions, agglutination assays (e.g., gelagglutination assays, hemagglutination assays), complement fixationassays, immunofluorescence assays, protein A assays, andimmunoelectrophoresis assays, etc.

The foregoing antibodies can be used in methods known in the artrelating to the localization and activity of the polypeptide, e.g., forWestern blotting, imaging the polypeptide in situ, measuring levelsthereof in appropriate physiological samples, etc. using any of thedetection techniques mentioned above or known in the art. Suchantibodies can also be used in assays for ligand binding, e.g., asdescribed in U.S. Pat. No. 5,679,582. Antibody binding generally occursmost readily under physiological conditions, e.g., pH of between about 7and 8, and physiological ionic strength. The presence of a carrierprotein in the buffer solutions stabilizes the assays. While there issome tolerance of perturbation of optimal conditions, e.g., increasingor decreasing ionic strength, temperature, or pH, or adding detergentsor chaotropic salts, such perturbations will decrease binding stability.

In a specific embodiment, antibodies that agonize the activity of thePPP1 protein can be generated. In particular, intracellular single chainFv antibodies can be used to regulate the PPP1 protein. Such antibodiescan be tested using the assays described below for identifying ligands.

In another specific embodiment, the antibodies of the present inventionare anti-idiotypic antibodies. These antibodies recognize and or bind toother antibodies present in the system. The anti-idiotypic antibodiesmay be monoclonal, polyclonal, chimeric, humanized.

In another specific embodiment, antibodies of the present invention areconjugated to a secondary component, such as, for example, a smallmolecule, polypeptide, or polynucleotide. The conjugation may beproduced through a chemical modification of the antibody, whichconjugates the antibody to the secondary component. The conjugatedantibody will allow for targeting of the secondary component, such as,for example, an antibiotic to the site of interest. The secondarycomponent may be of any size or length. In a specific embodiment, thesecondary component is a pharmaceutically active compound.

A further aspect of this invention relates to the use of antibodies, asdiscussed supra, for targeting a pharmaceutical compound. In thisembodiment, antibodies against the PPP1 protein are used to presentspecific compounds to infected sites. The compounds, preferably anantibiotic agent, when conjugated to the antibodies are referred to astargeted compounds or targeted agents. Methods for generating suchtarget compounds and agents are known in the art. Exemplary publicationson target compounds and their preparation are set forth in U.S. Pat.Nos. 5,053,934; 5,773,001; and 6,015,562.

EXAMPLES Materials and Methods

Bacterial Strains and Plasmids

S. pneumoniae strains utilized in this work were S. pneumoniae CP1200, anonencapsulated, highly transformable derivative of R36A, a roughvariant of D39, a virulent type 2 strain, (Morrison, D. A. et al., J.Bacteriology, 1983, 156:281) was obtained from Margaret Hostetter atYale University, C T., and S. pneumoniae strain 49136 obtained from theATCC. S. pneumoniae were grown to log phase (approx O.D. of 0.6–0.8 at600 nm) in Todd Hewitt media (DIFCO Lab., Detroit, Mich.) with 0.5%yeast extract (DIFCO) at 37° C. with aeration or on Tryptic Soy (DIFCO)blood agar plates. Escherichia coli strains used in this study wereBL21(DE3), BLR(DE3) (Novagen, Madison, Wis.), Top10F′(INVITROGEN, SanDiego, Calif.), and were grown in SOB media (15) at 37° C. with aerationcontaining appropriate antibiotics. Plasmids used in this work werePCR2.1 TOPO (INVITROGEN) and pET28a (Novagen). Where specified,chloramphenicol was used at 20 μg/ml, ampicillin at 100 μg/ml,streptomycin at 100 μg/ml, and kanamycin at 25 μg/ml. Restrictionenzymes were purchased from New England Biolabs (Beverly, Mass.) andused according to manufactures directions.

Identification of a Surface Associated Protein in Outer MembraneFractions of S. pneumoniae

Extraction of Surface Associated Components

Bacteria were grown in 4 liters of Todd Hewitt broth, and harvested bycentrifugation at 8000×g for 30 minutes. The pellet was suspended in˜175 ml of PBS with the aid of a pipette and immediately centrifuged at20000×g for 30 mm. The wash was filtered through a 0.45 m filter(NALGENE, Rochester, N.Y.), dialyzed and lyophilized.

Ion-exchange Chromatography of Surface Associated Protein Components

The PBS extract of S. pneumoniae was dissolved in Tris-HCl, pH 7.6 (10mM, 100 ml) and subjected to ion exchange chromatography in a column ofDEAE-SEPHAROSE CL-6B. After washing the column with the sample buffer,it was eluted first with 200 mM Tris-HCl, pH 7.6 followed by a linearNaCl gradient to a final NaCl concentration of 0.75 M (in 200 mMTris-HCl, pH 7.6) over 300 ml. Column fractions were analyzed bySDS-PAGE gel. Fractions containing a substantial amount of a surfaceassociated protein of approximately 18–20 kDa were pooled, desalted byCENTRICON SR3 concentrator and lyophilized.

N-terminal Amino Acid Sequence Analysis by PVDF Blot Excision.

The sample was diluted to 1 mg/mL total protein and combined 1:1 with2×Tris-SDS-β-ME sample loading buffer (0.25 M Tris-HCl pH6.8, 2% SDS,10% β-mercaptoethanol, 30% glycerol, 0.01% Bromophenol Blue) (OwlSeparation, Portsmouth, N.H.) and heated at 100° C. for 5 minutes.Approximately 10 μg of total protein (20 L of heated solution) of samplewas loaded in each of ten lanes on a 12 lane, 10 cm×10 cm×1 mm, 10–20%gradient acrylamide/bis-acrylamide gel (Zaxis, Hudson, Ohio). Molecularweight markers (Novex, San Diego, Calif.) were loaded in the twooutermost lanes of each side of the gel. Electrophoresis was carried outon an Owl Separations Mini-Gel rig at a constant amperage of 50 mA for 1hour in BIO-RAD Tris-Glycine-SDS running buffer. The gel was then rinsedwith deionized water and transferred to Millipore Immobilon-P PVDF(polyvinylidene fluoride) using a semi-dry blotting system supplied byOwl Separations at constant amperage of 150 mA for 1 hour. The resultingblot was stained with Amido Black (10% acetic acid, 0.1% amido black indeionized water) and destained in 10% acetic acid. The protein band wasthen excised from all ten lanes using a methanol cleaned scalpel ormini-Exacto knife and placed in the reaction cartridge of the AppliedBiosystems 477A Protein Sequencer (Foster City, Calif.). The N-terminalSequencer was then run under optimal blot conditions for 12 or morecycles (1 cycle Blank, 1 cycle Standard, and 10 or more cycles fordesired residue identification). PTH-amino acid detection was done onthe Applied Biosystems 120A PTH Analyzer. The cycles were collected bothon an analog chart recorder and digitally via the instrument software.N-terminal amino acid assignment was performed by comparison of theanalog and digital data to a standard set of PTH-amino acids and theirrespective retention times on the analyzer (cysteine residues aredestroyed during conversion and are not detected).

Subcloning and Expression of the Recombinant 20 kDa Surface AssociatedProteins

N-terminal sequence was compared against the NCBI non redundant databaselocated at www.ncbi.nlm.org using the BLAST algorithim developed byAltschul (Altschul, SF, et al., J. Mol-Biol., 1990, 215:403). Thisshowed that the N-terminal sequence had identity to a open reading frame(ORF) in NCBI database. This ORF had been previously sequenced and waslisted as an unidentified ORF (Pikis, A. et al., J. Infect. Dis., 1998,178:700). Subsequent BLAST analysis of the unknown ORF against thepublic release of the S. pneumoniae genome (serotype 4), made availableby The Institute for Genomic Research (TIGR, www.tigr.org), showed theORF to be present in the genome, but unidentified as well. DNA analysesof the unknown ORF in the S. pneumoniae genomic sequence and primerdesigns were performed using the DNASTAR (Madison, Wis.) Lasergene DNAand protein analysis software.

Primers flanking the ORF were designed (SEQ ID NOs: 1 and 2) andsubsequently synthesized using the ABI 380A DNA synthesizer. Tofacilitate subcloning the PCR product into the pET28a expression vector,restriction sites were designed into the PCR primers. An Nco1 site wasincluded in the 5′ primer, which allowed both for the ligation into theNco1 site of the expression vector and also included an ATG start codon.To maintain the correct reading frame, two extra bases were included inthe 5′ primer, resulting in the addition of a codon for Leucine. A Sal1site was included in the 3′ primer.

A PCR fragment of the expected size was generated from CP1200, ligatedinto the pCR2.1 vector, and used to transform ONE SHOT Top 10F′ cell(INVITROGEN). Ampicillin resistant transformants were screened byrestriction digestion of plasmid DNA prepared by alkaline lysis(Bimboim, H. C. and Duly, J., Nuc. Acid Res., 1978 7:1513). Arecombinant plasmid, containing the 20 kDa gene, was identified. DNAsequence was obtained from the clones using the Applied Biosystems PRISMDye Terminator cycle-sequencing core kit based on the PRISM protocolsupplied by the vendor. Approximately 1 ug of template DNA and 100 ng ofprimer were used for each cycling reaction. The reactions were cycled onthe GeneAmp® PCR Systems 2400 unit, purified using the PRISM method, andanalyzed on an ABI 373A DNA sequencer (Applied Biosystems).

The insert containing the r20 kDa gene was excised by restrictiondigestion with Nco1 and Sal1, and separated on a 1.5% Agarose gel. TheDNA fragment was cut from the gel and purified away from the agarose bya Bio 101 SPIN kit (Vista, Calif.). The insert was ligated with plasmidvector DNA(pET28a) also digested with Nco1 and Sal1, and wassubsequently transformed into Top 10F′ cells (INVITROGEN). The kanamycinresistant transformants were screened by restriction digestion ofplasmid DNA prepared by alkaline lysis (Bimboim, H. C. and Duly J., Nuc.Acid Res., 1978 7:1513). A recombinant plasmid was subsequentlytransformed into BL21 cells (Novagen) to create pLP533 and grown in SOBmedia supplemented with 30 ug/ml kanamycin. Cells were grown to anO.D.₆₀₀ of 0.6, and were subsequently induced with 0.4 mM IPTG(Boebringer Mannheim, Indianapolis, Ind.) for 2–4 hours. Whole celllysates were prepared and electrophoresed on a 15% SDS-PAGE gel(Laemmli, U. K., Nature, 1970,227:680) to confirm expression of thedesired recombinant product.

Purification of the Recombinant 20 kDa Surface Associated Protein.

A 250 mL flask containing 50 mL of SOB medium, supplemented with 30μg/ML kanamycin (Sigma, St. Louis, Mo.), was inoculated with a scrapingfrom a frozen culture of E. coli pLP533. The culture was incubated at37° C. with shaking at 200 rpm for approximately 16 hours. Subsequently,two 1 liter flasks containing SOB plus 30 ug/ml kanamycin wereinoculated with 20 mL of the overnight culture and incubated at 37° C.with shaking at 200 rpm. When the culture reached an optical density ofOD₆₀₀0.7–0.8, IPTG (Gold Biotechnology, St. Louis, Mo.) was added to 0.8mM. The culture was incubated at the same temperature with shaking foran additional three hours. The cells were then harvested bycentrifugation for 15 mm. at 7300×g. The cell pellets were frozen at−20° C. and were then thawed and resuspended in 300 mL of 10 mM sodiumphosphate pH 6.0 (J. T. Baker, Phillipsburg, Pa. ). The cell suspensionwas then passed through a microfluidizer (Microfluidics Corporation,Newton, Mass.) to lyse the cells. The lysate was centrifuged for 15 mm.at 16,000×g and the resulting supernatant was then centrifuged for 45mm. at 200,000×g Supernatants and pellets at each step were assayed bySDS-PAGE. The supernatant was diluted to 500 mL in 10 mM sodiumphosphate pH 6.0. The solution was then diafiltered with a 100,000 MWcutoff membrane (Millipore, Bedford, Mass.) against 1 L of the samebuffer and concentrated 2.5 fold. The protein, in the retentate, wasloaded onto a 70 mL ceramic hydroxyapatite column (BIO-RAD LaboratoriesHercules, Calif.) in 10 mM sodium phosphate pH 6.0. The column was thenwashed with 10 column volumes (CV) of the loading buffer. Contaminatingproteins were removed by washing the column with 10 CV of 108 mM sodiumphosphate pH 6.0. The protein was eluted from the column with a lineargradient over 10 CV from 108 mM to 500 mM sodium phosphate pH 6.0. Thepeak fractions were run on a 10% –20% SDS-PAGE gel (Zaxis, Hudson,Ohio). The fractions containing the protein were pooled and stored at−20° C. The protein was analyzed for homogeneity by SDS-PAGE, and theconcentration of protein during purification was determined by themethod of Lowry (Lowry, O. H., et al, S. Biol. Chem., 1951, 198:265).Protein concentration prior to immunization was determined using a BCAkit obtained from Pierce Chemicals (Northbrook, Ill.) and was usedaccording to the manufacturers directions. BSA was used as proteinstandard.

Polyclonal Antisera for Western Blot Analysis.

Recombinant protein was used to generate polyclonal antisera in mice.Briefly, 10 μg of r20 kDa protein was adjuvanted for each dose as anemulsion with Incomplete Freund's Adjuvant (IFA) (1:1v/v) and injectedsubcutaneously into 6–8 week old Swiss Webster mice. The mice were bledand vaccinated at wk 0, boosted at wk4, then exsanguinated at wk 6. Tenmice were vaccinated with the r20 kDa protein adjuvanted with IFA. Thesera were pooled and used for further analysis.

SDS-PAGE and Western Blotting.

Whole cell lysates were prepared by centrifuging equivalent numbers ofpneumococcal cells, based on the OD₆₀₀, in a microcentrifuge for 30 sec.Pneumococcal cell pellets were resuspended in an appropriate volume ofloading buffer. Where indicated, samples were boiled for 5 mm andseparated on a 10% SDS-PAGE gel using the method of Laemmli (Laemmli,Nature, 1970; 227:680). The samples were transferred to nitrocellulose(BioRad, Hercules, Calif.) using a BIO-RAD Mini Transblot cell (BIO-RAD)and the blots were blocked at room temp for 30 minutes in 5% nonfatmilk-PBS (BLOTTO). Pooled mouse antisera were used at a 1:1000 dilutionin BLOTTO for 60 minutes, followed by 25 minute washes in PBS-0.2% TWEEN80. Goat anti-mouse IgG+M conjugated to alkaline phosphatase (BiosourceInternational, Camarillo, Calif.) was used to detect bound antibodies ata 1:1000 dilution in BLOTTO. The blots were washed as previouslydescribed and detected with NBT and BCIP from BIO-RAD according to themanufacturer's directions.

Intranasal Immunization of Mice Prior to Challenge.

Six-week old, pathogen-free, Balb/c mice were purchased from JacksonLaboratories (Bar Harbor, Me.) and housed in cages under standardtemperature, humidity, and lighting conditions. BALB/C mice, at 10animals per group, were immunized with 5 μg of r20 kDa protein. On weeks0, 2, and 4. On each occasion, 5 μg r20 kDa formulated with 0.1 μg ofCT-E29H, a genetically modified cholera toxin that is reduced inenzymatic activity and toxicity (Tebbey, P. W., et al., Vaccine, 2000,18:2723), was slowly instilled into the nostril of each mouse in a 10 μlvolume. Mice immunized with Keyhole Limpet Hemocyanin (KLH)-CT-E29H wereused as controls. Serum samples were collected 4 days after the lastimmunization.

Mouse Intranasal Challenge Model.

Balb/c mice were challenged on week 4 day 6 with 1×10⁵ CFU's of serotype3 streptomycin resistant S. pneumoniae. Pneumococci were inoculated into3 ml of Todd-Hewitt broth containing 100 μg/ml of streptomycin. Theculture was grown at 37° C. until mid-log phase, then diluted to thedesired concentration with Todd-Hewitt broth and stored on ice untiluse. Each mouse was anesthetized with 1.2 mg of ketamine HCl (Fort DodgeLaboratory, Ft. Dodge, Iowa) by i.p. injection. The bacterial suspensionwas inoculated to the nostril of anesthetized mice (10 μl per mouse).The actual dose of bacteria administrated was confirmed by plate count.Four days after challenge, mice were sacrificed, the noses were removed,and homogenized in 3-ml sterile saline with a tissue homogenizer(Ultra-Turax T25, Janke & Kunkel Ika-Labortechnik, Staufen, Germany).The homogenate was 10-fold serially diluted in saline and plated onstreptomycin containing TSA plates. Fifty μl of blood collected 2 dayspost-challenge from each mouse was also plated on the same kind ofplates. Plates were incubated overnight at 37° C. and then colonies werecounted.

ELISA Assay for r20 kDa Protein.

Antibody titers against r20 kDa protein were determined by enzyme-linkedimmunosorbent assay (ELISA). ELISAs were performed using r20 kDa (100 μlper well of a 5 μg/ml stock in PBS, pH7.1) to coat Nunc-Immuno™ PolySorpPlates. Plates were coated overnight at 4° C. After blocking with 200 μlof PBS containing 5% nonfat dry milk (blocking buffer) for 1 hour atroom temperature, the plates were incubated with serial dilutions oftest sera diluted in blocking buffer for 1.5 hours at room temperature.The plates were then washed five times with PBS containing 0.1% TWEEN(PBS-T) and incubated with biotinylated goat anti-mouse IgG or IgA(1:8000 or 1:4000 in PBS; Brookwood Biomedical, Birmingham, Ala.) for 1hour at room temperature. After five additional washes with PBS-T, theplates were incubated with streptavidin conjugated horseradishperoxidase (1:10,000 in PBS; Zymed Laboratory Inc., San Francisco,Calif.) for 1 hour at room temperature. The plates were then washed fivetimes with PBS-T, incubated 20 minutes with 100 μl of ABTS substrate(KPL, Gaithersburg, Md.), followed by addition of 100 μl stoppingsolution (1% SDS). Absorbance values were read at 405 nm using aVERSAmax microplate reader (Molecular Devices Corp., Sunnyvale, Calif.).The end point titers of test sera were the reciprocal of the highestmean dilution that resulted in an OD₄₀₅ reading of 0.1. The meanbackground titers of test sera were quantified by absorbance values readat 405 nm on the wells that had all reagents except sera.

Statistical Methods. Comparison of nasal colonization among groups wasperformed using the Tukey-Kramer test (Ludbrook, J., Clin Exp PharmacolPhysiol., 1998, 25:1032). Results were considered significant at p<0.05.

Sequence Heterogeneity of PPP1.

To examine sequence heterogeneity for the PPP1 protein, the nucleotidesequence for the gene was compared among 10 different serotypes. GenomicDNA was prepared from overnight cultures of each serotype of S.pneumoniae. Cells were harvested by centrifugation at 1000×g for 15minutes at 4° C. and resuspended in 2 ml TE buffer. Cells were lysed bythe addition of SDS to 0.3% and Proteinase K (SIGMA) to 10 μg/ml. Thecells were incubated overnight at 55° C. Proteins were extracted fromthe cleared lysate by the addition of an equal volume ofphenol/chloroform/isoamyl alcohol (made by combining a 24:1 mixture ofchloroform/isoamyl alcohol with an equal volume of water saturatedphenol). The phases were separated by centrifugation at 7500×g for 10minutes at room temperature, then the aqueous phase was removed to a newtube. The process was repeated, then the DNA was precipitated from theaqueous phase by the addition of 10.4M NH₄Ac to 20%, and 2.5 volume ofethanol. The genomic DNA was spooled out using a glass rod andresuspended in 200 μl TE buffer. The gene for PPP1 was sequenced fromthe genomic DNA of serotypes 1,3,4,5,6,7,9,14,18,23F, and CP1200, usingthe Applied Biosystems Prism Dye Terminator cycle-sequencing core kitbased on the Prism protocol supplied by the vendor. Approximately 1 μgtemplate DNA and 100 ng of primers were used for each cycling reaction.The reactions were cycled on the Gene Amp PCR Systems 2400 unit,purified using the Prism method, and analyzed on an ABI 373A DNAsequencer (Applied Biosystems). The nucleotide sequences and theirpredicted amino acid sequences were aligned in the Megalign applicationof the DNA LASERGENE package from DNAstar, using the Clustal Walgorithm.

Evaluation of PPP1 Message Expressed in vivo.

Preparation of RNA from Cells Grown in vitro

Various S. pneumoniae serotypes were grown to log phase (O.D.₅₅₀ approx0.3) in 60 ml THB −0.5%YE at 37° C. with 5% CO₂. The cells wereharvested by centrifugation at 1000×g for 15 minutes at 4° C. Thesupernatant was aspirated and the cells were resuspended in 1 ml RNAselater (Ambion, Calif.) and stored for >1 hr at 4° C. The cells were thencentrifuged in a microfuge for 5 minutes at 8000×g. The supernatant wasaspirated and the cells were resuspended in 100 μl 10%Deoxycholate(DOC). 1100 μl of RNAZOL B (Tel-Test, Inc) was then added and thesuspension mixed briefly by inversion. 120 μl of CHCl₃ were then added,the sample mixed by inversion and then centrifuged in a microfuge atfull speed for 10 minutes at 4° C . The aqueous layer was removed andthe RNA was precipitated by addition of an equal volume of 2-propanol.The RNA was incubated at 4° C. for >1 hr and then centrifuged in amicrofuge at full speed for 10 minutes at room temperature. Thesupernatant was aspirated and the RNA was washed with 75% ethanol andrecentrifuged for 5 minutes. The supernatant was aspirated and the RNAwas resuspended in 50–100 μl nuclease free water. DNA was removed fromthe RNA by treating the sample with RNAse free DNAase (DNA FREE, Ambion)for 20 minutes at 37 degrees, followed by inactivation of the enzyme byaddition of the DNA FREE chleator. The purity and yield of the RNA wasassessed by measuring the absorbance at 260 and 280 nm.

Preparation of RNA from Cells Grown in vivo

Log phase S. pneumoniae cells were prepared as described above andresuspended to 106 cfu/ml in RPMI media (Celltech) supplemented with0.4% glucose. 1 ml of the cell suspension was sealed in a PVDF dialysismembrane with a 80,000 MW cutoff (SprectraPor). Two such bags wereimplanted intraperitoneally in 400 g Sprague Dawley rats. The bagsremained in the rats for 22 hours, after which the rats were terminatedand the bags were harvested. RNA was prepared from the intraperitoneallygrown cells as described above.

RT-PCR to Examine the Message for PPP1 in vivo

Message for the PPP1 gene was amplified out from both RNA prepared fromin vitro and in vivo grown cells using RT-PCR. A reverse PCR primercorresponding to the 3′ end of the gene was used to generate ds cDNA inthe following reaction. 1 μg RNA was incubated with 0.25 μM of thereverse primer: GGG GTC GAC TAA ACC AGG TGC TTG TCC AAG TTC (SEQ IDNO:8) for 3 minutes at 75° C., then cooled to 44° C. The message wasreverse transcribed using the RETROscript (Ambion) kit according to themanufacturer's directions. ReddyMix (ABgene) was used according to themanufacturer's directions to amplify the PPP1 message from 2–5 μl of thesample, using 0.25 μM of the above reverse primer and the forwardprimer: GGG GCC ATG GCT GTA GAA TTG AAA AAA GAA (SEQ ID NO:9). 10 μl ofthe amplified product was electrophoresed on a 2% Agarose gel.

Results

Identification of the 20 kDa surface associated protein—A PBS wash andion exchange chromatography was used to identify an 20 kDa surfaceassociated component of S. pneumoniae (FIG. 1). Lane 2–9 in FIG. 1represents fraction #8–16 from a DEAE column. There is clearly a majorprotein band between 15 and 20 kDa. The low molecular weight band wasresolved on a preparative SDS-PAGE gel and transferred to a PVDFmembrane. The PVDF membrane has a high binding capacity, which increasessample recovery and sequencing performance, allowing efficientdetermination of the amino terminal residues. The amino terminalsequence (SEQ ID NO: 3) of this protein allowed the identification of acorresponding open reading frame in the S. pneumoniae genome (SEQ IDNOS: 4 and 5). This ORF showed similarity to similar to non-hemeiron-containing ferritin proteins in other organisms, which may indicatesimilar function in S. pneumoniae (Pikis, A., et al., J. InfectiousDiseases, 1998, 178:700). However, the exact function and cellularlocation of the proteins in S. pneumoniae is unknown. Subcloning andexpression of this ORF provided recombinant material of the expectedsize (FIG. 2).Purification of the recombinant 20 kDa surface associated protein.Purification was aided by the solubility of the recombinant protein.Significant purification away from cellular membranes was achieved bysequential centrifugations. In addition, the characteristic oligomerformation was successfully utilized to remove the remaining lowmolecular weight contaminating proteins by diafiltration. The predictedcharge of the protein at neutral pH allowed the protein to be purifiedto greater than 90% homogeneity on a Hydroxyapatite column, as seen inFIG. 5.Reactivity of anti-r20 kDa surface associated protein sera. Polyclonalantisera to recombinant

20 kDa surface associated protein were generated in Swiss Webster miceto evaluate antigenic conservation of the protein among strains.Antisera to the r20 kDa protein reacted with proteins of approximately20, 40, and 80 kDa in unheated whole cell lysates of native species(FIG. 3), while the major reactive species seen in heated samples is atapproximately 20 kDa (not shown). These results suggest that thisprotein is part of a complex of 4 subunits or more.

Intranasal Challenge. To determine whether i.n. immunization with r20kDa surface associated protein can induce serum immune responses, Balb/cmice were administered 5 μg r20 kDa 3 times at biweekly intervals usingCT-E29H (0.1 μg/dose) as a mucosal adjuvant. Immune sera collected 4days after the last booster immunization were tested in theantigen-specific ELISA assays. At 4 days after the last boosterimmunization, strong, antigen-specific IgG and IgA antibody responseswere generated in mice immunized with r20 kDa- E29H (Table 6). Whencompared to the unrelated protein KLH, immunization with r20 kDa surfaceassociated protein was able to significantly reduce colonization of type3 S. pneumoniae the nasopharynx of BALB/C mice. (FIG. 4) The results arecomparable to the ability of the type 3 conjugate to reduce colonizationof the homologous serotype (Henrikson, J, et al. Alcohol Clin Exp Res,1997, 21:1630).

Antigen specific ELISA titers for r20kDa surface associated protein fromS. pneumoniae. Sera wk4d5 Sera wk4d5 Group IgG IgA 5 μg r20kDa + 0.1 ugCT-E29H 79,726 1563 5 μg Type-3-Conjugate + 0.1 ug CT-E29H <50 <50 5 μgKLH + 0.1 ug CT-E29H <50 <50 Note: Endpoint titers determined from poolsof 5 BALB/c miceSequence Alignment of the PPP1 protein from 10 serotypes. As shown inFIG. 6, the sequence of PPP1 is largely conserved among serotypes. Ascan be seen, serotype 9 is the most divergent serotype. The PP1 isolatedfrom this serotype showed 15 amino acid differences from the majority.The remaining serotypes showed less than 5 amino acid differences. Anoverall consensus sequence of PPP1 is shown in FIG. 6 (SEQ ID NO:20).RNA Amplification. A discrete band of the expected size is seen in boththe in vitro and in vivo samples (FIG. 7). The size of the product wasestimated to be full length by comparison to Hae III restrictionfragments of Lambda DNA.

The patents, applications, test methods, and publications mentionedherein are hereby incorporated by reference in their entireties.

Many variations of the present invention will suggest themselves tothose skilled in the art in light of the above detailed description. Allsuch obvious variations are within the full intended scope of theappended claims.

1. An immunogenic composition comprising an isolated Streptococcuspneumoniae Pneumo Protective Protein (PPP) comprising the amino acidsequence of SEQ ID NO: 5, wherein the PPP has a molecular weight ofabout 20 kDa and the ability to reduce colonization of pneumococcalbacteria.
 2. The immunogenic composition of claim 1 and apharmaceutically acceptable carrier.
 3. The immunogenic composition ofclaim 1, wherein said PPP has an isoelectric point of about 4.587. 4.The immunogenic composition of claim 1, wherein said PPP has a charge ofabout −14.214 at pH
 7. 5. The immunogenic composition of claim 2,wherein the composition further comprises at least one adjuvant.
 6. Theimmunogenic composition of claim 1, wherein the PPP is a recombinantprotein.
 7. The immunogenic composition of claim 1, wherein the PPP isisolated from S. pneumoniae.
 8. The immunogenic composition of claim 1,further comprising additional S. pneumoniae antigens.
 9. A method ofinducing an immune response in a mammal to Streptococcus pneumoniae PPP,said method comprising administering to said mammal an amount of theimmunogenic composition of claim 1 effective to induce said immuneresponse in said mammal.